Maite Martínez Aldaya

Christine Lors

Sandrine Salmon

tel. Jean-François F Ponge
email: jean-francois.ponge@wanadoo.fr

Avoidance bio-assays may help to test the ecological significance of soil pollution

Keywords: Avoidance, Ecotoxicological test, Folsomia candida, Soil pollution, Toxicity

Polluted soils are avoided by soil animals, a phenomenon which can be used as a cheap, sensitive tool for the early detection of environmental risk

Introduction

The repellence of toxic substances to soil animals has been known for a long time and was quantitatively assessed on several animal groups [START_REF] Eijsackers | Side effects of the herbicide 2,4,5-T affecting mobility and mortality of the springtail Onychiurus quadriocellatus Gisin (Collembola)[END_REF][START_REF] Hund-Rinke | Avoidance test with Eisenia fetida as indicator for the habitat function of soils: results of a laboratory comparison test[END_REF]. The ecological assessment of soil toxicity is currently achieved through population and bioaccumulation bio-assays [START_REF] Crouau | The use of Folsomia candida (Collembola, Isotomidae) in bioassays of waste[END_REF]. In standardized ecotoxicological tests using the earthworm Eisenia fetida (ISO 11268) and the springtail Folsomia candida (ISO 11267), populations of these soil animals are submitted to increasing doses of a toxicant, which they cannot escape during the duration of the bio-assay (one to three months). In nature, contrary to plants, most animals move to best places for feeding, reproduction and ecdysis, using chemical cues [START_REF] Bengtsson | Selective odor perception in the soil Collembola, Onychiurus armatus[END_REF][START_REF] Salmon | Earthworm excreta attract soil springtails: laboratory experiments on Heteromurus nitidus (Collembola: Entomobryidae)[END_REF]. They are also able to escape deleterious environment or food using the same cues [START_REF] Fábian | Short-term effects of the insecticide dimethoate on activity and spatial distribution of a soil inhabiting collembolan Folsomia fimetaria Linné (Collembola: Isotomidae)[END_REF], except when they are immediately killed or immobilized by high doses of a toxicant [START_REF] Best | Response of microarthropod populations to naphthalene in three contrasting habitats[END_REF]. Both attraction and repulsion are behavioural traits which increase the ecological fitness of sensitive species [START_REF] Tranvik | On the advantage of Folsomia fimetarioides over Isotomiella minor (Collembola) in a metal polluted soil[END_REF]. It has been demonstrated that most fungal strains and decomposition stages of leaf litter which were either attractive or repellent to soil springtails in the short-term were those which in the long-term favoured or disfavoured their growth, survival and reproduction, respectively (Sadaka-Laulan and [START_REF] Sadaka-Laulan | Influence of holm oak decomposition stage on the biology of Onychiurus sinensis Stach (Collembola: Onychiuridae)[END_REF].

From a functional point of view, a sound basis for the early risk assessment of soil pollution at the ecosystem level should be to test whether a given soil will allow or not a community of soil organisms to colonize it [START_REF] Filser | Experimental studies on the reactions of Collembola to copper contamination[END_REF], which lies on both dispersal to a habitat and population growth [START_REF] Marinissen | Colonization of new habitats by earthworms[END_REF]. It has been observed repeatedly that plant litter decomposition and building of a soil structure can be lost in polluted soils by the absence of key functional groups [START_REF] Gillet | Humus forms and metal pollution in soil[END_REF].

Such functional losses may result not only from direct or indirect toxicity of a soil but also from the repellence of the same soil towards potential colonizers [START_REF] Gillet | Are acid-tolerant Collembola able to colonise metal-polluted soil?[END_REF].

Recently, several papers pointed on the need to combine ecototoxicological tests with avoidance tests for a proper assessment of soil toxicity [START_REF] Heupel | Avoidance response of different collembolan species to Betanal[END_REF][START_REF] Greenslade | A comparison of Collembola species for toxicity testing of Australian soils[END_REF][START_REF] Da Luz | Avoidance tests with Collembola and earthworms as early screening tools for site-specific assessment of polluted soils[END_REF].

The present study was aimed at comparing short-term avoidance tests with long-term tests of toxicity, using a soil polluted by polycyclic aromatic hydrocarbons (PAHs). Folsomia candida (Willem), a soil-dwelling springtail (Collembola: Isotomidae) currently used for the laboratory assessment of soil toxicity [START_REF] Smit | Effects of soil type, prepercolation, and ageing on bioaccumulation and toxicity of zinc for the springtail Folsomia candida[END_REF], was considered representative of an invertebrate animal group which is widely distributed in the soil ecosystem and which plays a significant role in the regulation of microbial processes [START_REF] Petersen | A comparative analysis of soil fauna populations and their role in decomposition processes[END_REF]. Different strains of F. candida were compared, in order (i) to check for strain variation, given the high degree of polymorphism which is known to occur in this cosmopolitan species [START_REF] Goto | Variation in the mucro of Folsomia (Collembola: Isotomidae)[END_REF], (ii) to evaluate the sensitivity and validity of avoidance tests when performed on different clones of the same species [START_REF] Chenon | Genetic polymorphism in nine clones of a parthenogenetic collembolan used in ecotoxicological testing[END_REF].

Material and methods

The test soil used in our experiments was obtained from a former coke oven site in northern France (Nord-Pas-de-Calais) in September 2003, then kept frozen at the laboratory.

A control soil was collected in a nearby non-polluted, plant-restorated zone, within the same industrial site. Most physico-chemical properties of the test and the control soil are shown in Tables 1 and2. The moisture content was measured by gravimetry and the texture was determined by laser particle size analysis. Soil pH water was measured using a Consort® C83 pH-meter fitted with glass electrodes corrected for temperature and a Schott® box with Ingold® combined electrodes. Total organic carbon was deduced from total carbon and inorganic carbon values, which were determined with a TOC-5000A Shimatzu® analyser.

Total organic nitrogen was determined by the Kjeldahl method, and total phosphorus as well as metal concentrations (As, Cd, Co, Cr, Cu, Ni, Pb and Zn) were analysed by Inductive Coupled Plasma Atomic Emission Spectrometry (ICP-AES) in a 138 Ultrace Jobin Yvon® analyser after hot hydrofluoric and perchloric acid digestion of the solid phase.

Concentrations of the 16 PAHs of the US EPA list of compounds [START_REF] Greene | Protocols for Short-Term Toxicity Screening of Hazardous Waste Sites[END_REF] were measured using High Performance Liquid Chromatography in a 2690 HPLC Waters® analyser fitted with an ultraviolet inverted phase C 18 Supelco® columm (length 250 mm, internal diameter 2.1 m), coupled to a 996 Waters® UV photodiode array detector, after extraction by dichloromethane/acetone (50/50 v/v) using the Accelerated Solvent Extracter Dionex® ASE 200. Chemical analyses were made in triplicate.

The test soil was used pure and mixed with the control soil at 50, 10, 5, 1 and 0.35% concentration (w/w), just before each experiment run, after thawing overnight at 3°C. Deionized water was added in sufficient amount to obtain a paste for avoidance bio-assays or a solid substrate moistened at field capacity for population bio-assays. Rearing microcosms were checked weekly and deionized water was added to avoid desiccation. The use of a paste, instead of the natural soil, in avoidance experiments, was justified by the need to prevent animals from hiding in the soil when placed under constant illumination, and to achieve standard textural conditions of the substrate.

Five F. candida strains collected in France were compared. In all cases, the clones came from a single thelytokous (parthenogenetic) female which was collected from a soil sample after extraction of microarthropods in a Berlese funnel, then sorted live. The animals were reared on fine quartz sand moistened with tap water and they were fed ad libitum with finely ground cow dung which was free of pesticides and antibiotics. Strain 1 was collected in October 2002 in a neutral soil slightly polluted by hydrocarbons (soil over tar deposit in an abandoned oil refinery) at Pechelbronn (48°56'N, 7°50'E, pH water 6.7, ∑ 16 PAHs 6.0 mg.kg - 1 ). Strain 2 was collected at the same date in a neutral, unpolluted control soil, 15 m from the previous site (48°56'N, 7°50'E, pH water 7.0, ∑ 16 PAHs 0.9 mg.kg -1 ). Strain 3 was collected in May 2002 in an unpolluted neutral soil in the park of the laboratory (oak/hornbeam woodlot) at Brunoy (48°40'N, 2°30'E, pH water 7.7, ∑ 16 PAHs 1.2 mg.kg -1 ). Strain 4 was collected in October 2002 in an unpolluted acid soil at Pfaffenbronn (oak/beech woodland), five kilometres from the Pechelbronn site (48°59'N, 7°50'E, pH water 4.3, PAHs not determined).

Strain 5 was collected in May 2002 in an acid soil (oak/pine woodland) at Brunoy (48°40'N, 2°30'E, pH water 4.3, PAHs not determined). Previous to each experimental run all specimens used had never been into contact with the polluted soil nor with any other kind of pollutant.

Although the strains originated from very different sites, some of them were polluted, rearing conditions were standardized (quartz sand, moistened with tap water, with dry powdered cow dung added ad libitum), and the time between first inoculation by a female and experimental use of its offspring was such (at least a year) that we discarded any possible residual pollution in the rearing boxes. The test specimens were selected among fully developed (adult) animals of the same rearing box.

Avoidance experiments were performed in sterile polystyrene Petri dishes (55 mm diameter, 10 mm height), the bottom of which was lined with two half-disks of filter paper made of glass fiber (50 mm diameter). The entire surface of each disk was then covered with a soil paste. One control half disk was covered with the control soil, the other with the test soil or a mixture of both soils. The two half-disks were separated by a 2 mm space line, at the center of which one individual of F. candida was deposited. The position of the animal was recorded each 20 min up to 100 min. Previous assays showed that this duration was enough although necessary to let the animal chose definitely between both sides after preliminary exploration of the Petri dish. Twenty replicates, in two successive batches of ten, were followed together. During the experiment, Petri dishes were placed under a Sharp® fluorescent illuminator in a chamber at 20°C. Blank experiments using control soil at both sides checked for the absence of any light gradient effect which could bias the results [START_REF] Salmon | Responses to light in a soil-dwelling springtail[END_REF]. Totals of five counts over 100 min for each Petri dish were used for testing differences between control and polluted sides, using sign tests [START_REF] Sokal | Biometry[END_REF]. Notice that unit data, ranging from 0 to 5, measured the average position of a single individual (from nil to constant avoidance) during the duration of the experiment and thus were not replicated over time. Replication and corresponding calculation of the degrees of freedom concerned only the 20 petri dishes which were independently followed in the course of time. Previous experiments with the same design showed that average values for choice position should be preferred to end-point values, given that choice by Collembola is rapid, often occurring within 10 min, but erratic movements may still occur thereafter (Salmon andPonge, 1998, 2001;[START_REF] Sadaka-Laulan | Influence of holm oak decomposition stage on the biology of Onychiurus sinensis Stach (Collembola: Onychiuridae)[END_REF].

In the course of each avoidance experimental run, some animals were found dead or paralyzed, their number increasing with the concentration of the polluted soil in the dilution series. These animals were counted separately, then discarded from the data set used for the sign test.

Population response was assessed by introducing batches of ten adults into each of five rearing chambers (polystyrene boxes 45 mm diameter, 25 mm height), fifth-filled with the control soil or with the same mixtures of control/polluted soil as used for avoidance experiments. A small amount of dry cattle dung powder was added above the soil substrate, then boxes were incubated at 20°C in darkness during 40 days. At the end of the experiment, the whole population (including the ten females introduced at the start of the experiment) was collected. We used forceps for collecting animals which were visible over the substrate. Animals living deep in the substrate were collected by flotation under excess water, and all specimens were immediately counted. Population sizes were compared between control and test soils using t tests [START_REF] Sokal | Biometry[END_REF].

Results

The studied substrate exhibited a high content in PAH compounds but was not polluted by heavy metals, arsenic or cyanides (Tables 1 and2).

Figure 1 shows the results of all avoidance tests which were performed on the five strains of F. candida. There was no bias due to a possible light gradient effect, showing that light was homogeneously distributed throughout the observation area. All blank tests, using the control soil on both sides of Petri dishes, did not reveal any significant departure from random values, nor any mortality during the test period. At a test soil concentration of 10% or higher, specimens from all strains avoided the test side. Strain 4 (acid soil Pfaffenbronn) did not avoid the test side to the same extent than the other four strains, even at the highest concentration (100%), while avoidance was total for the other four strains. At 1% and 5% test soil, Strain 4 did not avoid the test side, while the other four strains did. At 0.35% test soil, avoidance was displayed by Strain 2 only. Thus the five strains could be classified in a decreasing order of sensitivity to the test soil: 2 > 1-3-5 > 4. For all strains, the decrease from the point of minimum (or nil) avoidance to the point of maximum (or total) avoidance was progressive.

The number of animals which were observed to die or become motionless during avoidance runs increased according to the concentration of the test soil (Fig. 2). All animals were alive and actively moving when the test soil was diluted more than 20 times (< 5%).

However, Strain 4 began to be affected only at 10% test soil, contrary to the four other strains, pointing again to a lower sensitivity of this strain.

Figure 3 shows the results of population tests. At 10, 50 and 100% test soil, all introduced animals were killed, whatever the strain. At 5% test soil, some animals were still alive in some boxes at the end of the experimental period (40 days), but reproduction was rarely observed. Below 5% test soil, reproduction occurred in all boxes, without any significant departure from the control soil. In the control soil the number of juveniles produced after 40 days was 119±30, 109±17, 39±5, 116±30 and 90±13 (mean±S.E.) with Strains 1 to 5, respectively. The reproduction rate and the coefficient of variation met the validity criterion of the ISO guideline (ISO 11267) except for Strain 3, which displayed a lower reproduction rate than all other strains (one-way ANOVA on log-transformed data followed by SNK procedure, P = 2.10 -4 ). Contrary to avoidance tests, threshold concentrations did not differ between strains and the passage from the absence of effect to total or near total effect was abrupt whatever the strain.

Discussion

Toxicity of the test soil to the Collembolan Folsomia candida was clearly shown by population tests (Fig. 3). When the test soil was diluted to 5% in the control soil the batch of 10 adult animals did not reproduce (strains 1 and 3) or even partially or totally died (other strains). At higher concentration, mortality at 40 days was total. The PAH content of the test soil could explain this toxicity, however, we cannot discard the effect of other compounds, not dosed in the present study, such as tar. A visual inspection of the polluted soil under a dissecting microscope revealed an abundance of dark more or less spherical pellets ≤ 1 mm which, when broken with scissors, revealed a black, vitrified core part surrounded by a mixture of organic matter and clay particles. Such strongly heterogeneous material may explain the absence of a linear, or even curvilinear dose-response relationship when the animals were let several weeks into contact with the test substrate. We hypothesize that there was a threshold of dilution above which the animals were able to avoid a few, dispersed toxic micro-sites and thus behaved like in an innocuous environment [START_REF] Tranvik | On the advantage of Folsomia fimetarioides over Isotomiella minor (Collembola) in a metal polluted soil[END_REF]. Such micro-heterogeneity of the substrate did not occur in our avoidance tests, probably because of the more intense mixing which resulted from the preparation of the muddy paste.

Depite the fact that the test soil was clearly toxic to F. candida and the control soil was not, we cannot discard possible effects of soil type to explain why the animals preferred the control soil in avoidance experiments. The use of another, not polluted soil, coming from the same industrial site, was preferred to a neutral substrate such as quartz sand. To be realistic an avoidance experiment should mimic what the animals have to their disposal in the site from which the test soil was originating, i.e. a patchwork of polluted and unpolluted micro-sites, reflected in ou test and control soils, respectively.

Our results point at a good correlation between avoidance and toxicity tests.

However, only avoidance tests discriminated between strains of F. candida. Genetic polymorphism has been demonstrated between clones of F. candida coming from several European laboratories but this could not be correlated with ecotoxicological responses to cadmium and phenanthrene [START_REF] Chenon | Genetic polymorphism in nine clones of a parthenogenetic collembolan used in ecotoxicological testing[END_REF]. In particular these authors showed that the lowest concentration affecting mortality and reproduction did not differ between strains, as in our study, but they did not perform avoidance experiments.

What is the ecological significance of pollution avoidance? In a study comparing two isotomid species coming from the Gusum area, a metal-contaminated site in Sweden, [START_REF] Tranvik | On the advantage of Folsomia fimetarioides over Isotomiella minor (Collembola) in a metal polluted soil[END_REF] observed that Folsomia fimatarioides was able to avoid diets and substrates highly polluted by heavy metals (Cu+Zn), while Isotomiella minor was not.

The former species was more abundant than the latter when exposed to the highest concentration of metals in the soil. The authors concluded that avoidance of polluted diets and substrates offered an advantage to F. fimetarioides over I. minor in polluted soils, because it allowed the former species to select less polluted micro-sites and food in a heterogeneous environment. This indicates that even tiny animals, such as springtails, are capable of perceiving differences in their habitat. At the scale of life of micro-arthropods, the landscape is made of islands of food resources and habitats between which the animals move and which they select for nutrition, moulting, and oviposition [START_REF] Joosse | Ecological aspects of aggregation in Collembola[END_REF]. Shape and colour are of no use within the soil where animals live in darkness, therefore they must use chemical and mechanical sense organs for detecting deleterious and attractive habitat and food. Species-specific aggregation in conditioned sites increases the probability to find suitable places and favours mating [START_REF] Joosse | Ecological aspects of aggregation in Collembola[END_REF]. Avoidance and attraction may be directional and selective [START_REF] Verhoef | Formation and ecological significance of aggregations in Collembola. An experimental study[END_REF]. Repellent substances may also act by discouraging food intake [START_REF] Fountain | Continuous monitoring of Folsomia candida (Insecta: Collembola) in a metal exposure test[END_REF] or by favouring non-directional dispersal [START_REF] Sjögren | Dispersal rates of Collembola in metal polluted soil[END_REF], thus increasing the chance an animal will find a suitable place where it will remain motionless [START_REF] Michelozzi | Eco-ethological analysis of the interaction between isoprene and the behaviour of Collembola[END_REF]. However, a high dose of pollutant or a longer time of exposure may decrease mobility [START_REF] Petersen | Effects of the insecticide dimethoate on the behaviour of Folsomia fimetaria (L.) (Collembola: Isotomidae)[END_REF]. Some animals became motionless in the course of our short-term avoidance test (Fig. 2). Thus, when the toxicant exhibits paralyzing effects, escape movements may prevent the animals from intoxication but only at some distance from the poison source. This is a key point for assessing the ecological significance of avoidance, in particular in the case of PAH mixtures, which act as narcotics and thus may inhibit locomotory activity [START_REF] Landrum | Bioaccumulation and critical body residue of PAHs in the amphipod, Diporeia spp.: additional evidence to support toxicity additivity for PAH mixtures[END_REF].

Pollution may affect soil animal communities and their functioning in two ways. First, toxicity kills animals or prevents them from feeding and reproduction. This only occurs at the right place where the pollutant has been deposited. Indirect effects through collapses in microbial and plant communities [START_REF] Gillet | Changes in species assemblages and diets of Collembola along a gradient of metal pollution[END_REF] or predator populations [START_REF] Abrahamsen | Effects of artificial acid rain and liming on soil organisms and the decomposition of organic matter[END_REF] are also spatially limited and fall in this category. Second, repellence of toxicants may help the animals to find refuges deeper in the soil or outside the pollution spot. In this case, the final result will depend on whether the animal is adapted to live in deeper soil or is able to move rapidly at the soil surface; thus it will depend on speciesspecific biological traits. Such spatial shifts have been described or they can be suspected from existing data. [START_REF] Gillet | Changes in species assemblages and diets of Collembola along a gradient of metal pollution[END_REF] observed that several surface-dwelling species of Collembola, collected in an organic soil strongly polluted by heavy metals (Zn+Cd), visited the underlying clay-rich mineral soil, and fed on the mineral substrate rather than on the metal-contaminated organic layer. Vertical migration of Collembola down to the mineral soil was experimentally demonstrated by [START_REF] Best | Response of microarthropod populations to naphthalene in three contrasting habitats[END_REF] following naphthalene application on the ground surface, but this phenomenon occurred only at the lowest rate of application. A decrease in horizontal migration was demonstrated at all rates of application. Thus the few existing data point to vertical migration of microarthropods to deeper soil layers rather than to outward horizontal migration. Consequences are that low rates of application of a pollutant, eventually not reaching toxic doses, may impoverish the topsoil in some animal species. This may result into functional shifts such as organic matter accumulation and proliferation of mycelial webs [START_REF] Gillet | Humus forms and metal pollution in soil[END_REF].

Whether avoidance tests should be preferred to mortality/repoduction tests for the risk assessment of soil pollution is prone to discussion. The laboratory assessment of repellence is cheaper (total duration of a test three to four hours, preparation and calculation included) and needs a much lesser amount of specimens than standardized ecotoxicological tests based on population response. Tests of a longer duration are even disqualified in the case of neurotoxic compounds, as mentioned above. From an ecological point of view, avoidance tests seem more relevant than classical ecotoxicological tests, and they are more sentitive to within-species populational differences. However, there are several limitations to a wider use of avoidance tests as an alternative to the direct assessment of toxicity. Some substances, such as cadmium salts, are not perceived as repellent, while they are toxic when the animals are forced to keep into contact with them [START_REF] Greenslade | A comparison of Collembola species for toxicity testing of Australian soils[END_REF]. This means that this toxicant cannot be avoided in the environment and, thus, will destroy entire populations, contrary to others which the animals currently escape. [START_REF] Sadaka-Laulan | Feeding preferences of the Collembolan Onychiurus sinensis for fungi colonizing holm oak litter (Quercu rotundifolia Lam.)[END_REF] showed that a basidiomycetous fungal strain was attractive to the collembolan Onychiurus sinensis in shortterm experiments while it affected negatively its growth, survival and reproduction in the longterm. However, examples of compounds or substrates which are both repellent and good for population growth are unknown.

The above examples show that a proper combination of avoidance and toxicity tests could be the best promising tool for the prediction of environmental effects of pollution. If avoidance occurs at the same or at a higher rate of application than toxicity, this means that pollution will definetely kill the populations of the test organism. If avoidance occurs at a lower rate of application than toxicity, as in our experiment, or if not any toxicity is detected while the pollutant or the substrate proves to be repellent, this means that pollution will deprive the site form the test organism even in the absence of toxic effects. Thus, avoidance can be used as a tool to detect environmental hazards other than toxicity.

Clearly, the proposed avoidance test, applying a toxic soil as an artifical paste at the bottom of a battery of Petri dishes is a compromise between the need for a rapid and easy assessment of soil toxicity, and the need to find more realistic and ecologically-relevant methods. Microscosms, filled with a natural test soil, allow avoidance to be studied at the community level [START_REF] Chauvat | Colonization of heavy metal-polluted soils by Collembola: preliminary experiments in compartmented boxes[END_REF][START_REF] Ponge | Interaction between humus form and herbicide toxicity to Collembola (Hexapoda)[END_REF][START_REF] Gillet | Are acid-tolerant Collembola able to colonise metal-polluted soil?[END_REF][START_REF] Garnier | Acid-tolerant Collembola cannot colonize metal-polluted soils at neutral pH[END_REF], and they can be at first sight considered more realistic. However, it needs several weeks for the animals to select between test and control substrates in a confined environment which, in the case of neurotoxic, volatile compounds acting at distance, may imped any assessment of behavioural effects. In this case, we consider more realistic to test the immediate reaction of the animals in a semi-natural system purposely designed for that.

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

 123 Fig. 1. Avoidance tests. Mean percent presence of five strains of Folsomia candida on polluted side of Petri dishes (5 counts at 20 min intervals, 20 replicates) at 6 dilution rates of the polluted soil. Differences between control and polluted side tested by sign test (N.S. = not significant; *, **, *** = significant at 0.05, 0.01 and 0.001 level, respectively). Control value is 50%

Table 1 .

 1 Physical and chemical characteristics of the test soil and the corresponding control soil. Means of three replicated measures followed by standard deviations. Concentrations are expressed on a dry soil basis

		Texture	pH water	Total organic carbon (%)	Total organic nitrogen (mg.kg -1 )	Total phosphorus (mg.kg -1 )	Σ 16 PAHs (mg.kg -1 )	Cyanides (mg.kg -1 )
	Test soil	Silty sand	7.9±0.02	9	1700	620	2894±54	0.8
	Control soil Medium silt 8.4±0.04	0.7	670	410	0.97±0.09	<0.1

Table 2 .

 2 Concentration of heavy metals (mg.kg -1 ) in the test soil compared with geochemical background, i.e. concentrations measured over a wide range of unpolluted agricultural and forest soils(Sterkeman et al. 2002). Means of three replicate measures were followed by standard deviations. Concentrations are expressed on a dry soil basis

Acknowledgements

The authors are indebted to the Agence de l'Environnement et la Maîtrise de l'Énergie (ADEME) for financial support and to the Agence Française de Normalisation (AFNOR) for fruitful discussions about the ecological significance of avoidance tests.


Hassan Cheaito
email: hassan.cheaito@insa-lyon.fr

Bruno Allard
email: bruno.allard@insa-lyon.fr

Joris Pallier
email: jpallier@centumadeneo.com

Paul Albouy

Guy Clerc
email: guy.clerc@univ-lyon1.fr

Pascal Pommier-Petit
email: ppommier-petit@centumadeneo.com

Design of a 35 kW Power Equipment for energy harvesting in the aeronautic context

Keywords: Energy Storage System, Battery Management System (BMS), Bidirectional converter (BDC), SiC MOSFET

Electrical power systems is steadily gaining in importance as more subsystems are electrified to improve overall aircraft efficiency and reduce CO2 emissions. This paper deals with the design of a 35 kW -2500 Wh equipment (SUNSET) intended to recover energy from aircraft electrical brake. First, the the main technical and electrical characteristics of SUNSET prototype are described. The design of the pack battery is detailed, highlighting the battery management. A power module has been designed and validated using Insulated Metal Substrate board. Losses and electrical performance are tested. From the system viewpoint, it is shown that the power density target is reached within the converter but still not satisfying at the storage system level.

I. INTRODUCTION

Electric Taxiing (ET) for landing systems will allow the aircraft to push-back and taxi under on-board generated electrical power without the main engines running [START_REF] Bataller-Planes | Power balance of a hybrid power source in a power plant for a small propulsion aircraft[END_REF]. The ET is estimated to save up to 4% of the total fuel used on a flight, especially for short range flights. In addition, this system allows aircrafts to push back without support from an external tug. Thus, ET equipped aircrafts will be able to operate more quickly, thereby reducing both gate and tarmac congestion and CO2 emission.

As part of the development of the more electrical aircraft, a new project of Storage energy UNit for Smart and Efficient operation on Tarmac (SUNSET) is being developed. The main objective of this project is to recover energy at braking during the aircraft taxiing phase (after landing). The final goal is to develop a demonstrator integrating both energy storage system and bidirectional converter. This paper describes the hardware design of the SUNSET equipment. It is important to note that this paper is a followup of the trade-off study presented in [START_REF] Cheaito | Preliminary Design of Energy Storage System and Bidirectional DC-DC Converter for Aircraft application[END_REF]. As mentioned in [START_REF] Cheaito | Preliminary Design of Energy Storage System and Bidirectional DC-DC Converter for Aircraft application[END_REF], the design of this equipment is very challenging because, in addition to the high power and energy densities (30 Wh/kg -2 kW/kg -60 Wh/dm 3 ), it should meet all the aeronautic specifications, in DO160 reference book, such as safety, vibration stress, electromagnetic compatibility (EMC), etc…. This paper is organized as follows. The functional architecture is reviewed in Section II. Section III covers the balancing strategy and the Energy Storage System (ESS). The DC-DC power converter is described in Section IV. Finally a few concluding remarks and further steps are drawn in Section V.

Synopsis of the architecture of the SUNSET prototype.

II. SYSTEM ARCHITECTURE

This section discusses the main sub-functions of the system in order to understand how the overall system will operate. As depicted in Fig. 1, the architecture is based on the following main sub-equipments:

• A control board,
• A power converter including drivers and IMS power board and inductors, • An energy storage system including battery cells, battery management system (BMS) and protection circuit, • Two interconnecting boards for LV and HV.

The control board consists in three main functions: the primary manager, digital control and ultimate fault monitoring. Besides, all functional states and communication are managed by the control board.

The bidirectional DC-DC converter is based on a buck architecture. The buck is used to lower the input HVDC voltage to the battery voltage level. On the other side, it is necessary to step-up the battery voltage to the input HVDC voltage level using a boost converter operation. The boost converter is obtained by replacing the diode of the buck converter by a second MOSFET.

The Energy Storage System (ESS) includes three independent packs of 100 cells. The cells are connected in series. In addition, the ESS contains one BMS per pack that monitors and balances the cell states of charge (SoC). Furthermore, a hardware isolation between the battery pack and the converter is a mandatory requirement. For this reason, a solid-state protection circuit (SSPC) board solution has been chosen for its compactness (compared to a circuit breaker). The SSPC limits the battery inrush current due to the charge of various capacitors. The SSPC is controlled by the BMS. The latter system disconnects the pack (by opening the SSPC) in case of a hard fault.

The J1 board manages the digital input and output signals between the SUNSET control unit and the aircraft one. The J2 board is the HVDC input of the SUNSET prototype which contains mainly the EMC filter.

A. Voltage Control

One of the intrinsic objectives of the SUNSET prototype is to maintain the aircraft HVDC network around a stable 560 V DC. In this Section, the different conditions to operate as a buck or boost converter are discussed. As mentionned before, the SUNSET prototype features two main operations: i. Charge or buck mode: it recovers the electrical energy supplied by the inverter during the electric brake phase.

ii. Discharge or boost mode: restore the electrical energy from the battery to the aircraft network through the inverter during electric traction phase.

Notice that there is a standby mode when there is no energy transferred between the battery and the aircraft network. Whatever the SoC of the battery pack, this mode is active when B < HVDC < C (see Fig. 2). However, to enter into buck or boost mode, there are two hysteretic conditions between the HVDC and battery pack voltages.

Buck: If HVDC > D the SUNSET prototype can recover energy from the aircraft network if and only if the SoC of the battery pack is lower than level F. The pack may be charged until level E. The standby mode will be reactivated when the HVDC voltage reaches level C.

Boost: If HVDC < A the SUNSET prototype can restore energy from the battery pack to the aircraft network if and only if the SoC of the battery pack is higher than level G. The battery pack may be discharged until level H. The standby mode will be reactivated when the HVDC voltage reaches level B.

For security issues, the point of level E might be limited to 90% of maximum charge level. On the other hand, to extend the battery lifetime, the point of level H might be limited to 20% of maximum charge level.

B. Balancing Strategy

Balancing in battery pack is the concern of literature focus about the hardware implementation [START_REF] Vitols | Design of an embedded battery management system with passive balancing[END_REF]. The most common topologies are divided into two categories:

• Passive: fixed shunt resistors or switched shunt resistors.

• Active: multiple switched capacitors, switched transformers and/or shared transformers. Balancing strategy is less highlighted in literature in the aeronautic context. This Section aims to discuss this issue. Indeed, the best strategy consists in optimizing the power capability: minimizing power losses and preventing cell overcharge. This is achieved by finding the optimal time and duration of cell power dissipation. With 100 cells in series the balancing is a fastidious task. In order to simplify the hardware implementation, the balancing method used in this study is based on the passive method: one resistor is connected across each cell of the pack to drain excess charge via a MOSFET [START_REF] Perişoarǎ | A Passive Battery Management System for Fast Balancing of Four LiFePO4 Cells[END_REF]. As a result, 100 resistors and 100 MOSFETs are integrated on the BMS board. Each group of cells is monitored by a slave BMS which belongs all to one master BMS.

The battery is made of LFP technology. The voltage as a function of the battery SoC is very flat [START_REF] Vitols | Efficiency of LiFePO4 battery and charger with passive balancing[END_REF]. That's why the open-circuit-voltage (OCV) of a cell is not so representative of the actual SoC. Thus, the strategy should be expressed directly in terms of SoC. In this paper, we consider that SoC is well-given from BMS. And will not be discussed for the sake of place. In order to summarize, two main algorithms exist to choose the appropriate cell states to balance. Top balancing: balance the cells at the fully charged state (and let them diverge at the bottom). Inversely, bottom balancing, balance the cells at the discharged state and let them diverge at the top. In fact, bottom balancing has no merit in systems that utilize dissipative balancing because energy is released from the highest capacity cell to align with the lowest capacity one without improving the run time. With top balancing, the majority of cells could have high SoC whereas only several cells have low ones. In that case, the majority will be discharged to reach the minimum charge level.

For these reasons, another strategy is proposed for continuous operation for all SoC level. It takes into account both the SoC average value among all cells and the unitary ones. This strategy is shown in the flowchart in Fig. 3. First, the average SoC among the 100 cells (defined as µ) is evaluated in real time. Then, the SoC of each cell (µi) is compared to the average. This strategy allows a variability (Δµi=µi-µ) between the cells' SoC. In the case of a low average SoC, there is no real need to balance very accurately the battery pack. For instance, at average SoC < 30%, 20% SoC variability is authorrized. However, the higher the average SoC, the more critical the variability value. Actually, at high average SoC, cells aging effect accelerates dramatically what compromises safety and security. This is why the SoC variability should not exceed 2% at the end of the battery pack charge. If not, cells could be overcharged leading to risk of thermal runaway. This algorithm will be implemented and validated in a further step.

III. BATTERY PACK

This Section describes how to choose the power and energy densities of cells in order to meet initial challenges.

A. A123system cell

To build the energy storage system (ESS), the mechanical design will be based on the smallest unit which is the cell. According to the trade-off in [START_REF] Cheaito | Preliminary Design of Energy Storage System and Bidirectional DC-DC Converter for Aircraft application[END_REF], the A123system cell (2.5 Ah) has shown a very competitive energy density (240 Wh/L and 108 Wh/kg).

B. Packaging

The mechanical design of the SUNSET equipment is optimized in terms of weight and volume. The design of the equipment is mainly based on printed circuit boards that include several functions, in order to reduce the wiring and increase compactness. 20 battery cells are packaged in series into one module (see Fig. 4). 5 modules in series constitute one pack. For each pack (100 cells in series), there is one BMS board and one SSPC board. The ESS of the SUNSET equipment comprises three packs. At each packaging level, the energy densities are evaluated. Fig. 5 shows how densities highly decrease from the cell level to the pack level. From these latter results, we should consider the degradation in energy densities. The volume density drops down to 70% of the theoretical value (240 to 69) whereas the mass density decreases to 37% (108 to 67). However, the energy still meets the target fixed at the beginning of the project.

C. Cooling design

The cooling system is designed based on and simulations from typical operation profiles and electrical losses in the devices at a specified ambient temperature (SiC MOSFET, battery cells and inductors). The mechanical design of sub-parts such as the battery module must reduce the local hot points in order to reduce the sizing of the cooling system. The cooling system is based on forced air ventilation (no water cooling authorized). The first results shown in Fig. 6 give an air flow requirement equal to 400m 3 /hr in order to limit the absolute temperature to 60°C.

IV. POWER CONVERTER This Section discusses the power converter architecture compatible with a nominal inrush power level of 35 kW.

A. Architecture

The designed DC-DC power converter is a two-voltagelevel converter [START_REF] Khan | A Family of High Efficiency Bidirectional DC-DC Converters Using Switching Cell Structure[END_REF]. A leg comprises two Insulated Metal Substrate (IMS) board and each switch is a two-parallel-chip MOSFET (Fig. 7). The first objective of this topology is to reduce voltage constraints on semiconductor devices what allows to lower the switching losses. Another objective is to minimize voltage constraints on magnetic devices as inductors and capacitors, for easier integration on PCB due to low form factors. As can be seen in Fig. 7, three battery branches are connected to a three-interleaved-phase converter. A 120° phase delay is managed between the three phases thus reducing conducted emissions. Indeed the first harmonic seen by the HVDC network is related to the number of phases. The switching frequency is chosen to set the first harmonic below EMC limits. The high side and low side transistors of both levels are triggered simultaneously by α and (1-α) respectively.

B. Design of a PCB

IMS board is increasingly used in power electronics for its excellent heat transfer properties. The IMS board also privileges SMD components for more compact designs. However, the IMS board has a strong influence on the EMC, namely at the routing level. Indeed, in one hand, the proximity of tracks with a large conductive surface will allow minimizing their inductive effects. On the other hand, the common-mode capacitance will be larger. Routing becomes a key point for getting a circuit with good performances. The floating node area must be as small as possible to limit the common-mode capacitance. The symmetry of the circuit is extremely important to avoid common-mode to differential-mode conversion [START_REF] Cheaito | Virtual Bulk Current Injection: Modeling EUT for Several Setups and Quantification of CM-To-DM Conversion[END_REF] (Fig. 8).

The SiC MOSFET technology seems to offer the best solution for high power density. The current design of the power converter based on this technology gives a final power density of 3kW/kg (including inductors and heatsink). Besides, to reduce EMI issues, kelvin connections are highly recommended. Leadless or low-lead components are preferred as well to reduce parasitic inductance. In fact, SMD MOSFETs provide an easier soldering and wiring from the industrial point of view. For these reasons, D²-PAK with 4 pins (SCTH90N65G2V) has been chosen for the application.

A preliminary switching unit (dashed square in Fig. 7) using IMS and SMD SiC technology has been designed and manufactured as shown in Fig. 8.

C. Validation test of the elementary power board

This Section discusses the electrical performances of the designed board (switching, losses, and nominal power).

1) Theoratical study

A theoretical analysis was able to predict the total losses in the high-side MOSFET of the board. It also allows evaluating losses distribution between switching (on and off) and conduction mode. For the sake of place, only the final results are shown, according to the measurement procedure detailed in [START_REF] Graovac | MOSFET Converter Losses[END_REF]. The conditions in Table 1 have been considered for the buck mode. Fig. 7 shows the distribution of losses in the high-side MOSFET (11.9 W).

2) Elerctrical study

Tests in boost-and buck-modes have been performed at the maximum power of 5.8 kW. The switching frequency is settled at 50 kHz and the duty cycle is 60%. The dead time is set to 500 ns. Only the results for the high-side MOSFET in buck mode are presented. The curves in Fig. 10 show VGS (yellow), VDS (green) and ID (red). This measurements validate the design of the IMS PCB. The gate voltage (VGS) does not exceed the minimum threshold of 1.7 V: no false turn-on will happen. Furthermore, the total losses have been evaluated using (1) from [START_REF] Lakkas | MOSFET power losses and how they affect powersupply efficiency[END_REF]:

𝑃 𝑡𝑜𝑡𝑎𝑙 = 𝑃 𝑐𝑜𝑛𝑑 + 𝑃 𝑐𝑜𝑚 + 𝑃 𝑟𝑟 + 𝑃 𝑔𝑎𝑡𝑒 (1)
Where: P cond : conduction losses P com : commutation losses P rr : reverse recovery losses

The total losses have been found to be around 11.5 W per MOSFET.

3) Thermal study

In order to validate the latter results, it was necessary to carry out thermal tests. First, the thermal resistance (Rth_IMS) is calculated for the IMS PCB and then with the heatsinks to determine the global Rth. A series of tests have been done using the MOSFETs in the saturation region operation (VGS <10V) to control the injected thermal power. The Rth can be deduced readily from the ratio of temperature elevation over the dissipated power. The Rth effective value is round 1.65°C/W for the overall board including the heatsink. Fig. 11 shows the elevation of temperature at IMS surface. Notice that the highest temperature point is 83°C when the ambient temperature is 28.3°C. Once Rth is given, the same switching conditions as in section IV.C.2 have been applied. Temperature elevation (ΔT) has been measured to be 20°C. This means that the power losses using thermal measurements are equal to 12.1 W (ΔT/Rth).

Table 2 compares the previous results using the three methods. A good agreement has been found between theoretical and experimental evaluations.

Theoretical

Electrical Thermal 11.9 W 11.5 W 12.1 W Table 2 Losses from the high side MOSFET on the elementary power board at 5.8kW

The MOSFETs on the elementary power board enables to transfer power with an effective efficiency of 99.6%. Main losses are then awaited form passive components.

V. CONCLUSION This paper has presented the main technical and electrical characteristics of the SUNSET equipment. A new balancing strategy has been proposed. It decreases gradually the variability regarding the cells'SoC in the battery pack. This work has shown that the power density of the cell alone is not significant. Study should take into account the packaging, BMS and protections. These added features reduce the densities to around 37% for the mass energy density and to 70% of the initial volume energy density.

IMS power switching unit has shown a good experimental agreement with the expected results. Six IMS boards are connected afterwards to create the entire power converter. The SUNSET demonstrator is under manufacturing. Next steps consist in validating of the performances of the whole equipment.

Fig. 1 .

 1 Fig. 1. The threshhold voltage on the HVDC and SoC on the battery pack to activate the buck or boost mode

Fig. 2 .

 2 Fig. 2. Flowchart of the balancing strategy dependent on the average SoC of the battery pack

Fig. 3 .Fig. 4 .

 34 Fig. 3. The 3D schematic of one module (20 cells in series) and one pack (5 modules in series)

Fig. 5 .

 5 Fig. 5. Thermal simulation of the module in typical operation condition.

Fig. 6 .

 6 Fig. 6. Architecture of the converter

Fig. 9 .

 9 Fig. 9. Main waveforms on the high-side MOSFET on the elementary board: VGS (yellow), VDS (green) and IDS (red)

Fig. 10 .

 10 Fig. 10. Infrared camera picture of the elementary power board at 5.8kW and 28°C ambient temperature.

Table 1

 1 Operating conditions of the elementary board Fig. 8. Losses breakdown in the high-side MOSFET on the elementary power board

	Switching Frequency	50 kHz	V input	325 V
	Duty cycle	0.6	I output	30.1 A
	Power	5.72 kW	Load	6.3

ACKNOWLEDGMENT

This project has received funding from the Clean Sky 2 Joint Undertaking under the European Union's Horizon 2020 research and innovation programme under grant agreement n°785585.* *This output reflects only the author's view and that the JU is not responsible for any use that may be made of the information it contains.


Keywords: Rec8, Cohesin, Aurora kinases, Oocytes, Meiosis, Separase, Bivalent segregation, Chromosome segregation, metaphase I, phosphorylation

 , whereas Rec8 at the centromere is protected from cleavage by the action of PP2A-B56 8-10 . However, in mammalian meiosis it is unknown whether Rec8 has to be equally phosphorylated for cleavage, and if so, the identity of the relevant kinase(s). This is due to technical challenges, as Rec8 is poorly conserved preventing a direct translation of the knowledge gained from model systems such as yeast and C. elegans to mammals. Additionally, there is no turn-over of Rec8 after cohesion establishment, preventing phosphomutant analysis of functional Rec8. To address the very basic question of whether Rec8 cleavage requires its phosphorylation in mammals, we adapted a biosensor detecting Separase activity to study Rec8 cleavage in single mouse oocytes by live imaging.

Crucially, through phosphomutant analysis we identified phosphorylation sites in Rec8 promoting cleavage. We found that Rec8 cleavage depends on Aurora B/C kinase activity, and identified an aminoacid residue that is phosphorylated in vivo. Accordingly, inhibition of Aurora B/C kinase during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation.

Results and discussion

Yeast Rec8 is phosphorylated by CK1 (Hrr25 in S. cerevisiae, Hhp2 in S. pombe) and DDK (Cdc7-Dbf4 in S. cerevisiae, possibly Hsk1/Dfp1 in S. pombe) for cleavage by Separase [START_REF] Katis | Rec8 phosphorylation by casein kinase 1 and Cdc7-Dbf4 kinase regulates cohesin cleavage by separase during meiosis[END_REF][START_REF] Rumpf | Casein kinase 1 is required for efficient removal of Rec8 during meiosis I[END_REF][START_REF] Ishiguro | Shugoshin-PP2A counteracts casein-kinase-1-dependent cleavage of Rec8 by separase[END_REF][START_REF] Le | The C-terminus of S. pombe DDK subunit Dfp1 is required for meiosis-specific transcription and cohesin cleavage[END_REF] . In C. elegans oocytes, Rec8 is phosphorylated by AIR-2 (Aurora B), which is recruited to the short chromosome arms in a Haspin kinase-dependent manner to bring about Rec8 cleavage [START_REF] Ferrandiz | Spatiotemporal regulation of Aurora B recruitment ensures release of cohesion during C. elegans oocyte meiosis[END_REF] . We hypothesized that also in vertebrate meiosis one of these kinases may be required for Rec8 cleavage.

Aurora kinases are primarily regulated through their localization and interaction with their molecular partners, and not their individual substrate specificities. In mammalian oocytes three Aurora kinases are expressed, namely Aurora A, B and the oocyte-specific Aurora C [START_REF] Nguyen | Specialize and Divide (Twice): Functions of Three Aurora Kinase Homologs in Mammalian Oocyte Meiotic Maturation[END_REF][START_REF] Marston | Multiple Duties for Spindle Assembly Checkpoint Kinases in Meiosis[END_REF] . They are required for proper bipolar spindle formation, chromosome alignment and correction of faulty attachments. To address whether chromosome segregation defects -indicative of failure to cleave Rec8-are observed upon loss of Aurora B and C, oocytes entering meiosis I were treated with inhibitors targeting both Aurora B and C, namely AZD1152 and ZM447439 [START_REF] Ditchfield | Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores[END_REF][START_REF] Mortlock | Discovery, synthesis, and in vivo activity of a new class of pyrazoloquinazolines as selective inhibitors of aurora B kinase[END_REF][START_REF] Balboula | Selective disruption of aurora C kinase reveals distinct functions from aurora B kinase during meiosis in mouse oocytes[END_REF][START_REF] Swain | Regulation of spindle and chromatin dynamics during early and late stages of oocyte maturation by aurora kinases[END_REF][START_REF] Sharif | The chromosome passenger complex is required for fidelity of chromosome transmission and cytokinesis in meiosis of mouse oocytes[END_REF] . Efficient Aurora B/C inhibition was verified by staining metaphase I chromosome spreads with a phospho-specific antibody against Histone H3pS (Serine) 10, a prominent substrate of both kinases also in oocytes [START_REF] Balboula | Selective disruption of aurora C kinase reveals distinct functions from aurora B kinase during meiosis in mouse oocytes[END_REF][START_REF] Swain | Regulation of spindle and chromatin dynamics during early and late stages of oocyte maturation by aurora kinases[END_REF][START_REF] Hirota | Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin[END_REF] (Figure 1A). Because of Aurora B/C's role in cytokinesis preventing polar body (PB) extrusion [START_REF] Swain | Regulation of spindle and chromatin dynamics during early and late stages of oocyte maturation by aurora kinases[END_REF][START_REF] Sharif | The chromosome passenger complex is required for fidelity of chromosome transmission and cytokinesis in meiosis of mouse oocytes[END_REF][START_REF] Vallot | Tension-Induced Error Correction and Not Kinetochore Attachment Status Activates the SAC in an Aurora-B/C-Dependent Manner in Oocytes[END_REF] , we verified whether these oocytes nevertheless underwent metaphase-to-anaphase transition, a condition to analyse chromosome segregation. Exogenously expressed Securin-YFP degradation was assessed by live imaging. As previously published, degradation occurred in an accelerated manner in oocytes without Aurora B/C activity due to the absence of error correction signaling to the Spindle Assembly Checkpoint (SAC) (Figure 1B) [START_REF] Sharif | The chromosome passenger complex is required for fidelity of chromosome transmission and cytokinesis in meiosis of mouse oocytes[END_REF][START_REF] Vallot | Tension-Induced Error Correction and Not Kinetochore Attachment Status Activates the SAC in an Aurora-B/C-Dependent Manner in Oocytes[END_REF] . Securin-YFP degradation shows that inhibitor-treated oocytes are not cell-cycle arrested and proceed through meiosis I.

To determine whether bivalent chromosomes segregate in absence of Aurora B/C kinase activity, chromosome spreads were prepared from metaphase II oocytes treated with Aurora B/C inhibitors throughout meiotic maturation. Whereas control oocytes harbored only paired sister chromatids (dyads), the majority of oocytes treated with Aurora B/C inhibitors contained several bivalents (paired chromosomes), indicating failures in meiosis I chromosome segregation (Figure 1C-G). This phenotype is not due to absence of error correction resulting in chromosomes being attached to only one pole, because -as we have shown previously-bivalents attached to a monopolar spindle still separate homologous chromosomes [START_REF] Gryaznova | Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II[END_REF] . Hence, Aurora B/C kinase may be involved in efficient arm cohesin removal in meiosis I. Indeed, bivalents in Aurora B/C inhibitor-treated oocytes maintain some Rec8 on chromosome arms, revealing failure of complete arm cohesin removal (Figure 1H).

Loss of Aurora B/C can be complemented in part by Aurora A [START_REF] Nguyen | Genetic Interactions between the Aurora Kinases Reveal New Requirements for AURKB and AURKC during Oocyte Meiosis[END_REF] , and complete loss of Aurora A interferes with proper spindle formation [START_REF] Saskova | Aurora kinase A controls meiosis I progression in mouse oocytes[END_REF][START_REF] Blengini | Aurora kinase A is essential for meiosis in mouse oocytes[END_REF] , leading to a cell cycle arrest in metaphase I [START_REF] Blengini | Aurora kinase A is essential for meiosis in mouse oocytes[END_REF] . To address whether Aurora A is also required for chromosome segregation in meiosis I, we treated oocytes with the Aurora A inhibitor MLN8237 [START_REF] Manfredi | Characterization of Alisertib (MLN8237), an investigational small-molecule inhibitor of aurora A kinase using novel in vivo pharmacodynamic assays[END_REF] from entry into meiosis I onwards. Such as Aurora A knock-out oocytes, MLN8237treated oocytes arrested in metaphase I, indicating that inhibition of Aurora A was efficient [START_REF] Saskova | Aurora kinase A controls meiosis I progression in mouse oocytes[END_REF][START_REF] Blengini | Aurora kinase A is essential for meiosis in mouse oocytes[END_REF] (Figure S1A). We added the Mps1 inhibitor Reversine to override the SAC arrest [START_REF] Touati | Mouse oocytes depend on BubR1 for proper chromosome segregation but not for prophase I arrest[END_REF] which is at least partially responsible for the metaphase I arrest induced by Aurora A inhibition [START_REF] Blengini | Aurora kinase A is essential for meiosis in mouse oocytes[END_REF] , and performed chromosome spreads 14 hours later. Figure S1A shows that inhibition of Aurora A and simultaneously overriding the SAC arrest rescued PB extrusion in 40 % of MLN8237 treated oocytes, similar to previously published data [START_REF] Blengini | Aurora kinase A is essential for meiosis in mouse oocytes[END_REF] . Importantly, MLN8237 treated oocytes extruding PBs also segregated chromosomes (Figure S1B).

Hence, we conclude that in presence of Aurora B/C, Aurora A is not required for cohesin removal in oocyte meiosis I.

It was attractive to speculate that Aurora B/C directly phosphorylate Rec8 to convert it into a Separase substrate. But it was just as likely that Aurora B/C activity were required for Separase activation itself.

Separase activity can be analysed in live cells using a biosensor containing the mitotic kleisin subunit Rad21 sandwiched between two fluorescent tags and fused to H2B, to localize the sensor to chromosomes for quantification [START_REF] Nikalayevich | Detection of Separase Activity Using a Cleavage Sensor in Live Mouse Oocytes[END_REF][START_REF] Shindo | Separase sensor reveals dual roles for separase coordinating cohesin cleavage and cdk1 inhibition[END_REF] . Of note, in oocytes expression of this and similar constructs containing kleisin subunits does not produce cohesive cohesin complexes, as those are put in place long before entry into the first meiotic division, before birth of the female [START_REF] Burkhardt | Chromosome Cohesion Established by Rec8-Cohesin in Fetal Oocytes Is Maintained without Detectable Turnover in Oocytes Arrested for Months in Mice[END_REF] . Loss of co-localization of the two fluorophores on chromosomes indicates Separase cleavage of the sensor (Figure 2A). Notably, sensor cleavage was not perturbed by addition of any of two Aurora B/C kinase inhibitors from resumption of meiosis I onwards (Figure 2B). Thus, Separase is activated with comparable efficiency in the presence or absence of Aurora B/C activity. Aurora B/C kinases are therefore not required for Separase activation in meiosis I.

To ask whether Aurora B/C targets Rec8 for cleavage by Separase, we decided to adapt the Rad21 sensor assay to Rec8. For this, a sensor construct containing full length Rec8 was cloned and in vivo cleavage by Separase was analysed by quantifying loss of co-localization of the two fluorophores in oocytes. A recent study had shown that a Rec8 sensor spanning 297-506aa is not cleaved in vivo, with the caveat that cleavage assays were performed in mitotic cells [START_REF] Maier | Separase cleaves the kinetochore protein Meikin at the meiosis I/II transition[END_REF] . In our hands, full-length Rec8 sensor was cleaved in vivo in meiosis I oocytes (Figure 3A). Importantly, cleavage was abolished when 39 out of 59 Serines and Threonines were mutated to Alanine (Figure 3A, Table S1), indicating that phosphorylation of at least some of those sites is required for Rec8 cleavage.

The low expression levels of the full-length sensor prompted us to test truncated versions of Rec8. Also, potential interference of other cohesin binding proteins when examining phosphorylation site mutations should be limited in truncated sensor constructs. Not surprisingly, a Rec8 fragment spanning the first 143aa and not containing any potential Separase cleavage sites was not cleaved at all in oocytes. A Rec8 fragment spanning 1-290aa and 143-290aa was not efficiently or not at all cleaved, respectively, even though they contain five potential minimal Separase cleavage sites, one of which allowed Rec8 cleavage in vitro in a previous study [START_REF] Kudo | Role of cleavage by separase of the Rec8 kleisin subunit of cohesin during mammalian meiosis I[END_REF] . The two most likely Separase cleavage sites (R434 and R454) [START_REF] Kudo | Role of cleavage by separase of the Rec8 kleisin subunit of cohesin during mammalian meiosis I[END_REF] are found in Rec8 fragment 380-486aa, and indeed, this fragment was efficiently cleaved (Figure 3B, S2A and B).

Cleavage was strongly reduced upon mutation of the two Separase cleavage sites (Rec8 380-486aa ExxD*2) (Figure 3B, Table S1). Accordingly, cleavage was Separase dependent, because it did not take place in conditional Separase knock-out oocytes [START_REF] Kudo | Resolution of chiasmata in oocytes requires separase-mediated proteolysis[END_REF] (Figure 3C, Figure S2C). Therefore, a biosensor containing a Rec8 fragment can be used to dissect the requirements for Separase cleavage in oocytes, unlike expression of a truncated sensor in mitotic cells, which needed to be fused with the protein Meikin to be cleaved in a recent study [START_REF] Maier | Separase cleaves the kinetochore protein Meikin at the meiosis I/II transition[END_REF] .

Cleavage of the C-terminal Rec8 380-486aa fragment was abolished when all 17 Serines and Threonines were mutated to Alanine, indicating that phosphorylation of at least some of these residues is necessary for efficient Separase cleavage (Table S1, Figure 3B). Furthermore, sensor cleavage did not occur in the presence of Aurora B/C inhibitor (Figure 3D). Mutating only 5 Serines to Alanine around and including a conserved Aurora B/C consensus site -Serine S482-was enough to completely abrogate cleavage of the sensor, and mutating three (S466, 467 and 470) or two Serines (S479 and 482) to Alanine significantly decreased cleavage (Figure 3E). Substitution of Serine 479 or 482 to Aspartate, mimicking a phosphorylated residue, allowed efficient cleavage, in agreement with these sites being phosphorylated.

However, we think that phosphorylation of other sites besides Serine 479 and 482 is required or takes place when these sites are mutated, because a fragment where 14 Serine/Threonine residues except in the Aurora B consensus at S482 (SRTS) were substituted to Alanine, is hardly cleaved (Figure 3F, compare to the vertical dotted lines representing standard deviations of the negative control). Crucially, phosphomimetic substitutions on Serine 479 and 482 allowed some cleavage of Rec8 380-486 14S/T-A, indicating that 1) phosphorylation of those sites (S479 and S482) does not take place in the 14S/T-A mutant and 2) phosphorylation of S479 and S482 promotes cleavage. Rec8 380-486aa peptide but not Rec8 380-486 17S/T-A produced in bacteria was thiophosphorylated by Aurora B-INCENP in vitro, indicating that Aurora B/C may indeed directly phosphorylate Rec8 (Figure 3G). Rec8 380-486aa sensor carrying 4 Aspartate substitutions around the conserved Aurora B/C consensus sites was cleaved in presence of Aurora B/C inhibitor, albeit with low efficiency, again indicating that other kinases and phosphorylation sites additionally contribute to Rec8 cleavage (Figure 3H). Altogether, our data strongly indicate that Aurora B/C-dependent phosphorylation of Rec8 contributes to Separase-dependent cleavage of Rec8, with Serine 479 and 482 being prominent, but not the only sites.

Next, we asked whether endogenous Rec8 is indeed phosphorylated in oocyte meiosis I in an Aurora B/C-dependent manner. In meiosis I, cohesin on chromosome arms is removed during the first division to allow separation of chromosomes, whereas a tiny fraction of cohesin in the centromere region is protected to maintain sister chromatids together until meiosis II [START_REF] Sato | Tell the Difference Between Mitosis and Meiosis: Interplay Between Chromosomes, Cytoskeleton, and Cell Cycle Regulation[END_REF][START_REF] Miller | Meiosis I: when chromosomes undergo extreme makeover[END_REF] . First, we wanted to determine the amount of endogenous centromeric Rec8 present in meiosis I and maintained until meiosis II. To address how much Rec8 is effectively protected in the centromere region, we stained endogenous Rec8 with a polyclonal antibody on chromosome spreads, obtained from meiosis I and II metaphase oocytes.

Kinetochores and underlying centromeres were stained with CREST serum. The Rec8 signal overlapping with CREST and in-between the two CREST signals was quantified and normalized against CREST. We found that less than 20% of Rec8 present between and overlapping with the two centromere signals is preserved from meiosis I to meiosis II (Figure 4A). In other words, 80 % of Rec8 is removed from this region already in meiosis I and may thus be phosphorylated, similar to Rec8 localized to arms. Hence, phospho-specific antibodies directed against the sites we identified should allow detection of phosphorylated Rec8 on chromosome arms, but not to distinguish phosphorylated from unphosphorylated Rec8 in the centromere region.

We produced phospho-specific antibodies in rabbits against the main sites identified with the Rec8 Separase sensor, namely S467, S470, S479, and S482 (see Figure S3A for scheme and peptide sequences). Aliquots of the sera were immunopurified against the phospho-peptides (alone or in pairwise combination). The sera were tested on Rec8 380-486aa sensor constructs expressed in oocyte meiosis I, either in wild type form or containing Alanine substitutions. S479 and S482 were recognized by the serum purified against those sites, but not when mutated to Alanine (Figure S3B andC). Sera directed against pS467 and pS470 did not recognize the sensor construct in its wild type form, and were thus not further used (Figure S3D).

Crucially, sera directed against pS482 and pS479-pS482 allowed detection of endogenous arm-localized cohesin in metaphase I, recognizable by the typical cross-shaped pattern that is usually observed when staining for Rec8 (Figure 4B). To assess presence of phosphorylated Rec8 on arms, we divided our spreads into three categories, as the intensity of the signal was highly variable, depending on the quality of the individual spreads, the degree of chromosome condensation and could therefore not be quantified (Figure 4C). As negative controls we used either pre-serum, or the serum pre-absorbed with phospho-peptide pS479-pS482 (Figure 4D). A prominent signal was also detected in the centromere region (Figure 4B). However, centromeres have the potential to generate unspecific background signals, with multiple Aurora kinase substrates being present there 8 that may cross-react with the serum we generated. To address this issue, we compared chromosome spreads from embryos at the 8-cell stage undergoing mitosis, with meiosis II chromosome spreads. Because Rec8 is meiosis-specific and substituted by Scc1 from the first mitosis onwards [START_REF] Tachibana-Konwalski | Rec8-containing cohesin maintains bivalents without turnover during the growing phase of mouse oocytes[END_REF] , this allowed us to distinguish the signal specific for Rec8 on paired sister chromatids in meiosis II from a background signal present at centromeres of paired sister chromatids in mitosis, on samples prepared exactly the same manner. Indeed, a strong and unspecific signal colocalizing with CREST was detected on mitotic spreads. In addition, a weak signal inbetween sister centromere dots exactly where centromeric cohesin is localized (Figure 4A), was detected in meiosis II (Figure S3E). However, this signal is expected to be buried beneath the unspecific signal on fused kinetochores in meiosis I 20 , preventing assessment of pRec8 staining in the centromere region in meiosis I. Together with the fact that the centromere region can be expected to contain both phosphorylated and unphosphorylated Rec8 (Figure 4A), we thus decided to limit our analysis to arm cohesin, as the pattern of cross-shaped staining is only observed for proteins of the cohesin complex.

The signal with Rec8 p482 antibody on arms is gone upon arm Rec8 cleavage in anaphase I, further indicating that the serum specifically recognizes Rec8 there (Figure S3F). When normalized to CREST, the background signal at centromeres did not decrease, indicating that the signal there is unspecific (Figure S3G). Thus, endogenous Rec8 on arms is phosphorylated on S482 (the Aurora B consensus site).

However, no conclusions concerning protected Rec8 in the centromere region can be drawn in meiosis I.

It is nonetheless interesting to note that pRec8 is detected between CREST dots in meiosis II (Figure S3E), suggesting that phosphorylation of S482 is involved in centromeric cohesin removal at anaphase II as well.

S482 corresponds to the minimum consensus site for Aurora B/C. If endogenous Rec8 were phosphorylated in an Aurora B/C-dependent manner, detection of arm-localized Rec8-p482 should fail in oocytes treated with Aurora B/C inhibitor, and this was indeed the case (Figure 4E). Total Rec8 staining was not affected by addition of ZM447439, showing that it is the phosphorylation of Rec8 and not Rec8 itself that is lost (Figure 4F). Together with the failure to correctly segregate chromosomes in meiosis I in absence of Aurora B/C activity we conclude that mouse Rec8 is phosphorylated in an Aurora B/C kinasedependent manner on S482 for Separase cleavage in meiosis I.

The Aurora B/C consensus motif we identified here in mouse oocytes can also be found in human Rec8, suggesting conservation of the mechanism for Rec8 cleavage in mammals. However, we think that also other sites are phosphorylated by Aurora B/C, and do not exclude potential roles for additional kinases, such as CK1 or DDK, which are required for Rec8 cleavage in yeast [START_REF] Katis | Rec8 phosphorylation by casein kinase 1 and Cdc7-Dbf4 kinase regulates cohesin cleavage by separase during meiosis[END_REF][START_REF] Rumpf | Casein kinase 1 is required for efficient removal of Rec8 during meiosis I[END_REF][START_REF] Ishiguro | Shugoshin-PP2A counteracts casein-kinase-1-dependent cleavage of Rec8 by separase[END_REF][START_REF] Le | The C-terminus of S. pombe DDK subunit Dfp1 is required for meiosis-specific transcription and cohesin cleavage[END_REF] . Aurora kinases have been shown to substitute for each other, and it is quite likely that in the absence of Aurora B/C, Aurora A can take over their role to a certain degree. This and the contribution of other kinases may explain why inhibition of Aurora B/C does not completely block bivalent segregation in meiosis I, and why meiosis II bivalents obtained upon Aurora B/C inhibition harbor only small amounts of Rec8 (Figure 1H). Aurora kinases function as catalytic subunits of the CPC (Chromosomal passenger complex) and have been shown to compete for CPC binding, with Aurora A having highest affinity, followed by Aurora B and C. Aurora C is localized to the interchromatin axis in a Haspin kinase-dependent manner, whereas Aurora B is restrained to the centromere region. It is attractive to speculate that because of its localization Aurora C is the major kinase required for Rec8 cleavage on arms in meiosis I. Indeed, an increase in active Aurora C-CPC complexes has been shown to cause precocious sister chromatid segregation in meiosis I, in agreement with Aurora C promoting cleavage [START_REF] Nguyen | Genetic Interactions between the Aurora Kinases Reveal New Requirements for AURKB and AURKC during Oocyte Meiosis[END_REF] .

We have discovered recently that an event we termed kinetochore individualization takes place in anaphase I and may require cleavage of centromeric Rec8 20 . However, we do not think that Aurora B/C dependent phosphorylation of Rec8 is required, because sister kinetochores still individualize in oocytes treated with Aurora B/C inhibitor (Figure 1D andE). Future work will aim at identifying how Rec8 accessibility is regulated, and the role phosphorylation plays in cleavage of distinct Rec8 fractions. (E) Chromosome spreads from mouse oocytes treated with 20 M Aurora B/C inhibitor ZM447439 from GVBD onwards, in metaphase II (GVBD+18h), stained for DNA (with Hoechst, white) and centromeres/kinetochores (with CREST antibody, red). Lower panels are an enlarged area as marked with a yellow dash line square and a schematic of a bivalent. On the schematic the chromosome arms are in blue and centromeres/kinetochores (individualized) are in red. Scale bar (white) is 10 m.

Main Figure legends

(F) Frequency of bivalent separation in meiosis I, quantified from chromosome spreads of metaphase II oocytes (GVBD+18h). Chromosomes were observed in control oocytes and oocytes treated with Aurora B/C inhibitors (10 m AZD1152 or 20 M ZM447439) and classified as "bivalents" (homologous chromosomes together in form of a bivalent), or "dyads" (homologous chromosomes separated into sister chromatid pairs). The number for dyads is divided by 2 to compare to the number of "not separated bivalents". n is the number of analyzed bivalents. *** p<0.0001, calculated with Fisher's exact test.

(G) Same data as (F), but numbers are per oocyte. Only groups of chromosomes for one oocyte in the same field were quantified. n is the number of oocytes. On each graph all frequencies are displayed as numbers on the corresponding bars. *** p<0.0001, calculated with Fisher's exact test. S1. F) Chromosome spreads prepared and stained as in e), except that a polyclonal Rec8 antibody was used.

Chromosomes with Rec8 localized on arms were quantified as in (E), according to the classification in (C).

A representative spread with a zoom on one chromosome is shown for each condition. n indicates the number of chromosomes analysed, scale bars are 10 m.

Related to Figure S3.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animals

Adult Swiss mice were purchased from Janvier, France, at 7 weeks of age and kept in the conventional animal facility of UMR7622 (authorization B75-05-13) in an enriched environment and with food and water access ad libitum. Separase -/-mice and litter mate control mice (C57BL/6-Tg) were house-bred in the animal facility of UMR7622. The local ethical committee (comité d'éthique en experimentation animale Charles Darwin, no 5, Ile-de-France) authorized the project (registered under 03263.02), in accordance with national guidelines and regulations. Female mice were sacrificed by cervical dislocation between 8-16 weeks of age, ovaries were dissected and GV stage oocytes harvested. Mice were not involved in any procedures before sacrifice except for genotyping, and hormonal stimulation to obtain embryos.

METHOD DETAILS

Mouse oocyte and embryo culture

Oocytes were harvested in M2 medium (homemade or Merck Millipore, MR-015P) and kept arrested at GV stage with 100µg/mL dbcAMP (dibutyryl cyclic AMP, Sigma-Aldrich, D0260). After release in dbc-AMP free M2 medium, oocytes undergoing GVBD no later than 90min after release were used for experiments. Oocytes were cultured in M2 medium drops covered by mineral oil, manipulated on heating plates and kept in a temperature-controlled incubator at 38°C. For longer incubation, oocytes were cultured in M16 medium (MERK Millipore MR-10P or homemade) in a CO 2 -and temperaturecontrolled incubator. To obtain 8-cell stage embryos in mitosis, superovulation was induced in female mice by intraperitoneal injection of PMSG (pregnant mare serum gonadotropin, 5 IU/female) and hCG (human chorionic gonadotropin, 5 IU/female) 48 hours later. Then, females were coupled with a male, verified by the presence of a vaginal plug. Pregnant female mice were sacrificed 12h after fertilization, embryos collected and incubated in EmbryoMax CZB medium (Sigma-Aldrich) with 1mM supplementary L-glutamine (Sigma-Aldrich) until they reached 8-cell stage, then incubated with 4 µM Nocodazole (Sigma Aldrich) for 6 hours to keep the cells arrested in the 4th mitosis.

Drug treatments

All inhibitors used were dissolved in DMSO (Sigma-Aldrich, D2650) and diluted to M2 or M16 medium at the indicated final concentrations. For Aurora B/C inhibition, ZM447439 (Tocris, 2458) was used at 20 µM, ZM447439 (Santa Cruz, sc-200696) was used at 2-20 µM and AZD1152 (Sigma Aldrich, SML0268) at 10µM. Cytokinesis failure but no metaphase I arrest was used as a positive control for only Aurora B/C (and not Aurora A) inhibition in every experiment. Optimal concentrations had to be determined for every lot of ZM447439. For Aurora A inhibition, MLN8237 (Bertin Bio-reagent, 13602) was used at 1µM, and metaphase I arrest was used in every experiment as control. To bypass the SAC, Mps1 inhibitor Reversine (Cayman Chemical, 10004412) was used at 0,5 µM, and also added to the oil covering the culture droplets. Acceleration of meiosis I was verified as control for efficient Mps1 inhibition.

Plasmids

Plasmids used are described in Table S1. Rad21 sensor construct was a gift from O. Stemmann (University of Bayreuth). Cloning of the plasmids was carried out using restriction enzymes (Thermo Fisher), PfuI polymerase (Promega) for PCR, and T4 ligation kit (Thermo Fisher). DNA was purified with QIAprep Spin Miniprep Kit, QIAquick PCR purification kit or QIAquick Gel Extraction kit (Qiagen). Cloning strategies for each construct are described in Table S1. All primer sequences are listed in Table S2. All primers were synthesized commercially (Eurofins). After cloning, plasmid sequences were verified through a commercial sequencing service (GATC).

Production of truncated GST-Rec8 proteins

Rec8 380-486aa and Rec8 380-486aa 17S/T-A were PCR cloned into pGEX-6P-2 (Primer sequences available upon request). Expression of GST-Rec8 proteins was induced in BL21(DE3)pLys bacteria (Promega, L1195) with 0.5 mM isopropyl-β-d-thiogalactopyranoside (Euromedex, EU0008-B) for 3 h at 37°C. After centrifugation at 10,000 × g for 15 min at 4°C, bacteria were lysed in PBS buffer supplemented with 1 mM PhenylMethylSulfonyl fluoride (Roche 39212624) in the presence of detergents (0.1% Triton X100 and 0.1% NP40) and sonicated. After ultracentrifugation (100,000 × g, 4°C, 1 h), the supernatant containing recombinant GST-Rec8 proteins was loaded for 1h30 at 4°C on 1ml glutathione-agarose beads (Sigma G4510), previously equilibrated in PBS buffer. After washing, proteins were eluted with PBS and 10 mM glutathione. The purified proteins were dialyzed overnight against PBS buffer and frozen at -80°C for further use.

in vitro Kinase assays

In vitro thiophosphorylation [START_REF] Allen | A semisynthetic epitope for kinase substrates[END_REF] of Rec8 proteins, either WT-Rec8 or 17S-A-Rec8, was performed by incubating 165 ng of recombinant GST-Rec8 proteins with 120 ng of recombinant Aurora B-INCENP (MERCK, item#14-835) in 30 µl of kinase buffer (40 mM MOPS, pH 7.3; 100mM MgCl2, 1mM EDTA; 0,1% -Mercaptoethanol) supplemented with 5mM ATPS for 90 min at 30°C. Each reaction was incubated for 30 mins at RT with 12.5 mM p-nitrobenzyl mesylate (Abcam, ab138910) for alkylation and the reaction was stopped by adding Laemmli buffer. Incorporated thiophosphates (P) were immunoblotted using an antibody directed against alkylated thiophosphates (Abcam, ab92570, 1:2000). Membranes were stripped twice for 15 mins at RT in 0.1M Glycine pH2.2 supplemented with 1% SDS and then probed using a HRP-GST antibody (Sigma, 16-209, 1:5000). Western blots are representative of three independent experiments.

Generation of phospho-specific antibodies

Using the services of Covalab (France), 2 rabbits each were primed with three peptides against each phospho-epitope, respectively: pSELpS (S467 S470): pSELSLEAAEDEKSR, SELpSLEAAEDEKSR, pSELpSLEAAEDEKSR; and pSRTpS (S479 S482): EDEKSRTpSLIPPEWWA, EDEKpSRTSLIPPEWWA, EDEKpSRTpSLIPPEWWA (Covalab). Sera of the rabbits with the highest titer were immunopurified against the individual peptides according to instructions by Covalab, and used as indicated. To detect sensor fragments, spreads were performed on metaphase I spreads and the sera were diluted 1:40, and to detect endogenous Rec8, spreads were performed on prometaphase I spreads and sera diluted 1:20 and 1:50.

Microinjection

mRNA coding for Securin-YFP [START_REF] Niault | Changing Mad2 levels affects chromosome segregation and spindle assembly checkpoint control in female mouse meiosis I[END_REF] , or sensor constructs was transcribed using the mMessage Machine T3 or T7 Kit (Invitrogen) and purified with RNeasy mini-kit (Qiagen). GV oocytes were injected on an inverted Nikon Eclipse Ti microscope with self-made micropipettes (Narishige magnetic puller, PN-31) and a FemtoJet microinjector pump (Eppendorf) with constant flow. Oocytes were manipulated using a holding pipette (vacuTip from Eppendorf).

Chromosome spreads

After removal of the zona pellucida by successive transfer in acidic tyrode solution, chromosomes were spread by transferring oocytes in hypotonic solution (3 mM DTT, 0,15% Triton-X100, 1% Paraformaldehyde), placed on 8 wells glass slides(Fisher Scientific, 10588681) [START_REF] Chambon | Chromosome spreads with centromere staining in mouse oocytes[END_REF] . Upon drying at room temperature, chromosome spreads were stored at -20°C until immunostaining. Thawed spreads were washed several times with PBS 1X, incubated 10 min with PBS-BSA 3%, and incubated with primary antibodies diluted in PBS-BSA 3% for 2-3h. The following antibodies were used at the indicated concentrations: Human CREST serum (Immunovision, HCT-100, at 1:50 or 1:100), Rabbit poly clonal anti-Rec8 (gift from Scott Keeney, 1:50 and 1:100), Rabbit anti-pRec8 (directed against the indicated phospho-peptides, 1:20, 1:40, 1:50), anti-phospho-Histone H3 (pS10H3, 1/100 dilution; Merck Millipore, 05-806). To detect endogenous Rec8 efficiently, spreads had to be performed in prometaphase I (GVBD+ 4-5 hours), before complete chromosome condensation. Spreads were washed several times with PBS and incubated with the following secondary antibodies diluted in PBS-BSA 3%, for at least 1 hour: donkey DNA was stained with Hoechst 33342 (Invitrogen, H21492, 50µg/mL). After several PBS washing steps chromosome spreads were mounted in Vectashield (Eurobio H-1000) or AF1 mounting medium (CityFluor) and stored at 4°C until acquisitions with a spinning disk confocal microscope.

Image Acquisitions

Live imaging of oocytes expressing Securin-YFP was performed on a Nikon eclipseTE 2000-E inverted microscope coupled to a Prime sCMOS camera, equipped with PrecisExite High Power LED Fluorescence, a Märzhäuser Scanning Stage and using a Plan APO (20x/0,75NA) objective. Time lapse was set up for 6h with 20 min intervals. 1 z-section was acquired for every time point in the GFP channel and in DIC. Live imaging of oocytes expressing sensor constructs was performed on a Zeiss Axiovert 200M inverted spinning disk confocal microscope, coupled to an EMCCD camera (Evolve 512, Photometrics), using a Plan-APO (63x/1,4 NA) oil objective. Time lapse was set up for 10 hours with 20 min intervals. For each timepoint, 11 z-stacks (3 µm optical section spacing) were acquired in 491nm channel for GFP or YFP and in 561nm channel for mCherry. Only oocytes expressing comparable leves of sensor constructs and undergoing metaphase-to-anaphase transition without delay were further analysed. All live imaging was done under perfectly temperature-controlled conditions, and microscopes were piloted by Metamorph software. Fixed chromosome spreads were acquired on the same Zeiss Axiovert 200M inverted spinning disk confocal microscope, but using a 100x/1.4 NA oil objective. 6 z sections of 0,4 µm interval were taken in each channel. Images were processed using Fiji software. For publication, images were treated in Adobe Photoshop to adjust brightness and contrast, all adjustments were applied to whole images uniformly, and to all images being compared.

QUANTIFICATION AND STATISTICAL ANALYSIS

Quantifications were performed with Fiji software. For quantification on chromosome spreads, the mean fluorescence intensities were measured on sum-projected images. A square is drawn around centromeres as depicted on the scheme in Figure 4A or on chromosome arm. The measured signal is corrected to background. Data plots and statistical analysis were obtained with Graphpad PRISM 8 software. Quantifications of cleavage efficiencies of different sensors were done as described [START_REF] Nikalayevich | Detection of Separase Activity Using a Cleavage Sensor in Live Mouse Oocytes[END_REF] . In brief, mean fluorescence signal for YFP (or GFP) and mCherry was measured in the box surrounding chromosomes during several timepoints before, during and after anaphase I, corrected for background fluorescence. The ratio between YFP (or GFP) and mCherry signals was calculated and the difference between pre-anaphase value (normalized to 100%) and first post-anaphase value derived. All experiments were done at least three times independently, the total number of oocytes analysed in each condition is indicated.

Figure 1 Aurora

 1 Figure 1 Aurora B/C activity is required for homologous chromosome separation in meiosis I. (A) Chromosome spreads from mouse oocytes in prometaphase I (GVBD+4h) stained for DNA (with Hoechst, blue), centromeres (with CREST antibody, red), and Histone H3 phosphorylated on Ser10 (H3pS10, green). Scale bar (white) is 10 m. The graph on the right shows the corresponding quantification of H3pS10 fluorescence intensity detected on chromosome arms. Each dot is a measurement from one chromosome arm corrected for background. Bars and whiskers are means and SD. (B) Accelerated Securin-YFP degradation in oocytes treated with AZD1152 or ZM447439. Securin-YFP fluorescence intensity in the cytoplasm was measured in time lapse images of mouse oocytes undergoing maturation. n is the number of oocytes. Hours after Germinal Vesicle Breakdown (GVBD) are indicated. Time points were taken every 20 minutes. Each dot and whiskers (shaded areas) are means and SD.(C) Chromosome spreads from mouse oocytes in prometaphase I (GVBD+4h) and metaphase II (GVBD+18h), stained for DNA (with Hoechst, grey) and centromeres/kinetochores (CREST antibody, red).Lower panels are enlarged areas marked with yellow dash line squares and a schematic of a bivalent (left) and a sister chromatid pair (right). On the schematic the chromosome arms are in blue and centromeres/kinetochores are in red. Scale bar (white) is 10 m.(D) Chromosome spreads from mouse oocytes treated with 10 M Aurora B/C inhibitor AZD1152 from GVBD onwards, in prometaphase I (GVBD+4h) and metaphase II (GVBD+18h), stained for DNA (with Hoechst, grey) and centromeres (with CREST antibody, red). Lower panels are enlarged areas marked with yellow dash line squares and a schematic of bivalents. On the schematic the chromosome arms are in blue and centromeres/kinetochores are in red. Of note, centromeres/kinetochores are separated on meiosis II bivalents due to kinetochore individualization. Scale bar (white) is 10 m.

( H )

 H Chromosome spreads such as in (E) and stained with polyclonal Rec8 antibody. ZM447439 was added from GVBD onwards. Related to Figure S1.

Figure 2 Aurora

 2 Figure 2 Aurora B/C activity does not inhibit Separase-dependent cleavage of Rad21 sensor. A) Schematic of sensor construct (top), example of time lapse microscopy acquisition of a mouse oocyte expressing the Rad21 sensor, +/-40 minutes (min) relative to anaphase I onset (middle) and an example of the quantification of GFP/Cherry ratio (on the right). Anaphase I is labeled as 0 minutes timepoint. Scale bar (white) is 20 m. Cleavage of the sensor is visible by the disappearance of the GFP signal from the chromosomes, while the Cherry signal remains. Quantification of the GFP/Cherry ratio measures mean signal from the area occupied by the chromosomes corrected for the background signal from the cytoplasm. Sensor cleavage efficiency is quantified as per cent decrease of the GFP/Cherry ratio when comparing the last timepoint before anaphase I onset and the lowest point during anaphase I. B) Cleavage of Rad21 sensor in control oocytes and oocytes treated with the indicated inhibitors of Aurora B/C. Shown are examples of time lapse microscopy images of mouse oocytes expressing the Rad21 sensor, +/-40 minutes relative to onset of anaphase I. Scale bar (white) is 20 m. C) Quantification of cleavage efficiency of Rad21 sensor from experiment in (B). n is the number of oocytes analysed. Bars and whiskers are mean and SD. n.s. -not significant, according to Mann-Whitney test.

Figure 3 CleavageF)

 3 Figure 3 Cleavage efficiencies of Rec8 sensor constructs in oocyte meiosis I. The sensor constructs that were analysed are indicated on the left with corresponding schemes, and the corresponding in vivo cleavage efficiencies when expressed in oocytes, are on the right. Negative control (ctrl) indicates a sensor construct without Rec8 sequence and no cleavage site. In the schemes, potential Separase cleavage sites are indicated in green, single amino acid substitutions in red, multiple amino acid substitutions indicated in grey, and the relative position of the Rec8 fragment being tested is shown by its position on the black line (not in scale). On each graph n is the number of oocytes, bar and whiskers are means and SD. Vertical dotted lines mark standard deviation of the negative control sensor. n.s.not significant, * p<0.01, ** p<0.001, *** p<0.0001 according to t test performed for each condition compared to negative control. A) Cleavage of sensor construct containing full-length Rec8, and Rec8 harboring 39 Serine or Threonine to Alanine substitutions. B) Cleavage efficiencies of four Rec8 fragments (1-143aa, 143-290aa, 1-290aa, 380-486aa), and Rec8 380-486aa harboring mutations in two Separase cleavage sites, or 17 Serine or Threonine mutated to Alanine. C) Cleavage efficiencies of Rec8 380-486aa sensor construct in Separase control (Sep +/+ ) and Separase knock-out (Sep -/-) oocytes. *** p<0.0001 according to t test comparing Sep-/-to Sep+/+. D) Cleavage efficiencies of Rec8 380-486aa sensor construct with or without being exposed to Aurora B/C inhibitor AZD1152 from GVBD onwards. *** p<0.0001 according to t test comparing inhibited oocytes to control. E) Cleavage efficiencies of Rec8 380-486aa harboring Alanine substitutions in two, three and five potential phosphorylation sites (5S-A: S466, 467, 470, 479, 482A; 3S-A: S466, 467, 470A; and 2S-A: S479, 482A) or Aspartate substitutions (SRTD: S482D; DRTS: S479D). S482 corresponds to an Aurora B consensus site. * p<0.01, *** p<0.0001 according to t test performed for each condition compared to negative control. F) Cleavage efficiencies of Rec8 380-486aa harboring 14 Alanine substitutions except Serine 479 and 482, which were either Serine (SRTS) or Aspartate (DRTD). ** p<0.001 according to t test comparing SRTS to DRTD. G) In vitro kinase assay using bacterially expressed GST-Rec8 380-486aa (WT) and GST-Rec8 380-486aa 17S/T-A (A) as substrates for GST-Aurora B-INCENP. Above: incorporation of P into Rec8 and Aurora B

Figure 4 Phospho

 4 Figure 4 Phospho-specific antibodies recognize Rec8 in an Aurora B/C kinase dependent manner. A) Oocytes in metaphase I and metaphase II were fixed for chromosome spreads right next to each other, in the same wells, and stained with polyclonal Rec8 antibody (magenta), anti-CREST serum (centromeres/kinetochores, green), and Hoechst (DNA, in blue). A representative spread and a zoom on one bivalent are shown for each condition on the left. n indicates the number of oocytes analysed, scale bars are 10 m. Below, quantifications of Rec8 signal overlapping with CREST, or inbetween the two CREST signals, as indicated in the scheme. *** p<0.0001, by Mann-Whitney Test. B) Chromosome spreads of oocytes in prometaphase I were stained with the indicated sera to detect endogenous Rec8 phosphorylated on S482, or both S479 and S482 (magenta), together with anti-CREST serum (centromeres/kinetochores, green), and Hoechst (DNA, in blue). A representative spread is shown for each condition. n indicates the number of oocytes analysed, scale bars are 10 m. C) Spreads stained with phospho-specific anti-Rec8 antibodies were classified according to the examples of stainings shown in (B). D) Serum specific for pS479pS482 was pre-absorbed with peptide pS479 pS482, and used to stain chromosome spreads as described in (B). The results of quantifications according to the classification in c) are shown. E) Chromosome spreads of control prometaphase I oocytes and oocytes treated with ZM447439 from GVBD onwards, were stained with serum directed against pS482 to detect endogenous Rec8 phosphorylated on S482 (magenta), together with anti-CREST serum (centromeres/kinetochores, green), and Hoechst (DNA, in blue). The graph shows quantifications of the indicated number of chromosomes, according to classification in (C). A representative spread with a zoom on one chromosome is shown for each condition. n indicates the number of oocytes analysed, scale bars are 10 m.

  anti-human Cy3 (709-166-149, Jackson Immuno Research, 1:200), donkey anti-Human Alexa Fluor 488 (709-546-149, Jackson Immuno Research, 1:200), donkey anti-Rabbit Cy3 (711-166-152, Jackson Immuno Research, 1:200), donkey anti-Rabbit Alexa Fluor 488 ( 711-546-152, Jackson Immuno Research, 1:200).

Chromosome segregation in human oocytes is highly error prone, one reason being weakening of cohesin complexes with age, causing high rates of pregnancy loss 33 . Insights into the molecular mechanisms of cohesin removal will be crucial to better understand how these errors occur.

Acknowledgements

We thank members of the MOM team for discussion and helpful suggestions, Eileen Breunig for preliminary experiments, Olaf Stemmann for Rad21 sensor construct, mRec8-39A plasmid and hosting EN in his lab for several months, Kim Nasmyth for the Separase conditional knock-out mouse strain, Scott Keeney for Rec8 polyclonal antibody and Léa Lemaire (Covalab) for useful advice on the generation of phospho-specific antibodies. EN was supported by a postdoctoral fellowship by the FRM (Fondation de la Recherche Médicale SPF20150934093) and EMBO (short term fellowship ASTF 426 -2015). The KW lab obtained funding for this project by ANR (ANR-16-CE92-0007-01, ANR-19-CE13-0015), FRM (Equipe FRM DEQ 20160334921, Equipe FRM DEQ 202103012574), and core funding by the CNRS and Sorbonne Université.

DATA AND CODE AVAILABILITY

All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code.

Author contributions

Sensor assays were mostly done by EN, antibody stainings by SEJ and SAT with participation of CF and EN. EB contributed to Figure 1, SAT did sensor assays in Figure S2

Declaration of interests

The authors declare that they have no conflict of interest.

STAR Methods

LEAD CONTACT

Further information and requests for resources and reagents should be directed and will be fulfilled by the lead contact, Katja Wassmann (katja.wassmann@upmc.fr)

MATERIALS AVAILABILITY

All unique reagents generated in this study are available from the lead contact without restriction. The study did not generate new mouse lines.

Table S1

List of Rec8 sensor constructs used in this manuscript. Related to STAR Methods.