Fanny Beauche ´¤

Freddie-Jeanne Richard
email: freddie.jeanne.richard@univ-poitiers.fr

The Best Timing of Mate Search in Armadillidium vulgare (Isopoda, Oniscidea)

Mate choice is mediated by many components with the criteria varying across the animal kingdom. Chemical cues used for mate attractiveness can also reflect mate quality. Regarding the gregarious species Armadillidium vulgare (isopod crustacean), we tested whether individuals can discriminate conspecifics at two different levels (between sex and physiological status) based on olfactory perception. Tested conspecifics were individuals of the same or opposite sex, with the females at different moult stages. We found that the attractiveness of individuals was mediated by short-distance chemical cues and tested individuals were able to discriminate and prefer individuals of the opposite sex. Moreover, male preference to female increased during their moulting status as they matured. Males were particularly more attracted by females with appearing white calcium plates, which corresponds to the beginning of their higher receptivity period. These differences in attractiveness due to sex and physiological status are likely to shape the composition of aggregates and facilitate mate finding and optimize the reproductive success for both males and females. Thus aggregation pheromones could be linked to sex pheromones in terrestrial isopods.

Introduction

Mate Finding and Cues

As mates are not always receptive, mate finding should not include only locating a mate but also doing so with good timing. Indeed, the ability to discriminate receptive from unreceptive females prevents males from carrying out costly courtship unnecessarily [START_REF] Johansson | The role of chemical communication in mate choice[END_REF]. Individuals emit signals attractive to mates using different channels. Attractive cues can also be used as a phenotype of mate quality and can be visual [START_REF] Green | Flock formation and the role of plumage coloration in Ardeidae[END_REF], acoustic [START_REF] Takacs | Communication ecology of webbing clothes moth: attractiveness and characterization of male-produced sonic aggregation signals[END_REF], tactile [START_REF] Cullen | Behavioural phase change in the Australian plague locust, Chortoicetes terminifera, is triggered by tactile stimulation of the antennae[END_REF], or chemical [START_REF] Candolin | The use of multiple cues in mate choice[END_REF]. Male chemical profiles communicate much information on its quality: symmetry, parasite/pathogen load, mate recognition and the quality of individual immune system ( [START_REF] Ali | Female spotted cucumber beetles use own cuticular hydrocarbon signature to choose immunocompatible mates[END_REF][START_REF] Blomquist | Cuticular lipids[END_REF] and references therein). Female chemical compounds can also be responsible for inducing male courtship behavior [START_REF] Ginzel | Z9-pentacosen contact sex pheromone of the longhorned beetle Megacyllene robinae[END_REF]. Sex pheromones that have been studied are indicative of mate location or receptivity but are also used for mate assessment [START_REF] Candolin | The use of multiple cues in mate choice[END_REF].

Signaling their receptivity can also be beneficial for females in order to insure fertilization as well as to prevent male harassment when they are unreceptive. Higher attractiveness of receptive females have been observed in various species [START_REF] Garcia | Relationship between sexual interactions and the timing of the fertile phase in captive female Japanese macaques (Macaca fuscata)[END_REF][START_REF] Guevara-Fiore | Male mate-searching strategies and female cues: how do male guppies find receptive females?[END_REF][START_REF] Head | Male southern water skinks (Eulamprus heatwolei) use both visual and chemical cues to detect female sexual receptivity[END_REF][START_REF] Roberts | Information content of female chemical signals in the wolf spider, Schizocosa ocreata : male discrimination of reproductive state and receptivity[END_REF]. Female receptivity is often signaled through chemical cues as in the snapping shrimp Alpheus angulatus where males are able to detect females approaching their receptivity period through distance chemical communication [START_REF] Mathews | Tests of the mate-guarding hypothesis for social monogamy: male snapping shrimp prefer to associate with high-value females[END_REF]. This can be expected as changes of physiological and reproductive status generally imply modifications of internal chemistry [START_REF] Johansson | The role of chemical communication in mate choice[END_REF].

In crustaceans in particular, courtship behaviors can be very diverse from absent to very sophisticated. Mate choice can be the result of the female emitting signals attractive for males [START_REF] Krang | The ability of the amphipod Corophium volutator (Pallas) to follow chemical signals from con-specifics[END_REF] or the opposite in other species [START_REF] Popper | Acoustic detection and communication by decapods crustaceans[END_REF] or mate encounter can occur just by chance [START_REF] Thiel | The behavioral ecology of crustaceans: a primer in tasonomy, morphology and biology[END_REF]. Moreover, sexual pheromones and the impact of moulting cycles on females attractiveness have been reported in numerous crustacean species [START_REF] Chang | The crustacean endocrine system and pleiotropic chemical messengers[END_REF]. Mating and fertilization are closely associated with the female moulting cycle.

Aggregation and Mate Choice

Spatial and temporal distribution of mates can impact mate choice and individual dispersion to find non-relative mates. Inbreeding avoidance is a major determinant of animal dispersal and mating patterns [START_REF] Koenig | Reproductive roles in the cooperatively breeding acorn woodpecker: incest avoidance versus reproductive competition[END_REF] although surprisingly in some species, it appears to be absent [START_REF] Bennett | Reproductive suppression in subordinate, non-breeding female Damaraland mole-rats: two components to a lifetime of socially induced infertility[END_REF]. Gregarious species present a high risk of mating with relatives due to increased social interactions and attractiveness of conspecifics through aggregation pheromones [START_REF] Bartelt | Aggregation pheromones in five taxa of the Drosophila virilis species group[END_REF][START_REF] Bloom | A sexual aggregating pheromone system in the zebrafish, Brachydanio rerio (hamilton buchanan)[END_REF][START_REF] Graves | Snake aggregation pheromones: source and chemosensory mediation in western ribbon snakes (Thamnophis proximus)[END_REF][START_REF] Rivault | Cuticular extracts inducing aggregation in the German cockroach, Blattella germanica (L.)[END_REF][START_REF] Siljander | Identification of the airborne aggregation pheromone of the common bed bug Cimex lectularius[END_REF]. However, mate choice for inbreeding avoidance has been described in the gregarious Blattella germanica [START_REF] Lihoreau | Mutual mate choice: when it pays both sexes to avoid inbreeding[END_REF].

In Oniscidea, aggregation is one of the numerous traits resulting from the adaptation of Crustaceans from aquatic to terrestrial life (brood pouch, conglobation posture…) [START_REF] Hornung | Evolutionary adaptation of Oniscidean isopods to terrestrial life: structure, physiology and behavior[END_REF][START_REF] Smigel | Conglobation in the Pill Bug, Armadillidium vulgare, as a water conservation mechanism[END_REF]. In terrestrial isopods, the aggregation pheromone is excreted via the faeces [START_REF] Takeda | Aggregation phenomenon in terrestrial isopods[END_REF]. In the species Ligia exotica, Porcellionides pruinosus and Armadillidium vulgare, aggregation was shown to limit desiccation as well as to reduce metabolic rate and accelerate body growth [START_REF] Takeda | Aggregation phenomenon in terrestrial isopods[END_REF]. The level of aggregation depends on the level of humidity in the environment and varies with the geographic location of populations [START_REF] Takeda | Aggregation phenomenon in terrestrial isopods[END_REF][START_REF] Caubet | Geographical variability of aggregation in terrestrial isopods: What is the actual significance of such behaviour[END_REF] but it is always at its highest during the period of reproduction. This co-occurrence suggests a role of aggregation in mate finding. Furthermore, aggregation directly affects reproduc-tion because when females are in groups, and particularly with males, female vitellogenesis is accelerated [START_REF] Caubet | Biotic triggers of female reproduction in the terrestrial isopod Armadillidium vulgare Latr[END_REF][START_REF] Lefebvre | On the male-effect in the terrestrial crustacean Armadillidium vulgare (Latreille, 1804)[END_REF][START_REF] Lefebvre | Female-extended control over their reproductive investment: the role of early mating interactions on oocyte maturation in the terrestrial crustacean Armadillidium vulgare (Latreille, 1804)[END_REF] while female reproductive activity is synchronized through chemical cues transmitted via faeces [START_REF] Mead | Belated and decreased reproduction in isolated females of Helleria brevicornis Ebner (Crustacea, Oniscoidea). Recuperation after the addition of faeces to the female environment[END_REF]. In Oniscus asellus, female sizes affect their probability of being courted but mating success is independent of female and male size [START_REF] Stange | What renders a female Oniscus asellus attractive to a soliciting male? A priming approach[END_REF].

Moulting and Reproductive Cycle

Female attractiveness is linked to their moulting status in many crustacean species [START_REF] Chang | The crustacean endocrine system and pleiotropic chemical messengers[END_REF]. In terrestrial isopods, female moulting and reproductive cycles are closely linked [START_REF] Steel | Mechanisms of coordination between moulting and reproduction in terrestrial isopod Crustacea[END_REF] and have been extensively studied. The secretory cycle of the Y-organ (also called moulting gland) corresponds to the moulting cycle and initiates pre-ecdysis [START_REF] Charmantier | Contro ˆle endocrine et neuroendocrine de la mue de puberte ´chez les ma ˆles de Sphaeroma serratum (Fabricius, 1787) (Crustacea, Isopoda, Flabellifera)[END_REF]. Changes in the concentration of ecdysteroid secreted in the hemolymph mediate biochemical and physiological processes that occur during the moulting cycle [START_REF] Chang | Regulation of crustacean molting: A review and our perspectives[END_REF]. Females can experience two types of moult: normal when nonbreeding or parturial during the period of reproduction (respectively NM and PM in fig. 1). Indeed, the marsupium (brood pouch), necessary for egg incubation, only differentiates during these parturial moults. Moreover, complete ovarian maturation is only achieved during preparturial or parturial intermoult (PPM and PM in fig. 1) while during normal intermoult (NM in fig. 1) only the first stage of vitellogenesis occurs. The moulting cycle can be divided into five stages as described by Drach and Tchernigovtzeff [START_REF] Drach | Sur la me ´thode de de ´termination des stades d'intermue et son application ge ´ne ´rale aux Crustace ´s[END_REF] and adapted to terrestrial isopods by Steel [START_REF] Steel | Stages of the intermoult cycle in the terrestrial isopod Oniscus asellus and their relation to biphasic cuticle secretion[END_REF]: i) A/B period or post-ecdysis represents about two days following the moult when animals neither move nor eat and are vulnerable to predation and desiccation; ii) C period or di-ecdysis is the period of maximal activity and it is also the period of incubation for breeding females; iii) D period or pre-ecdysis is the period during which ovarian maturation as well as hormonal and physiological changes occur. Indeed, calcium is stored in the form of white plates of calcium carbonate which begin to appear at the stage D 1 ([39,40]; see fig. 1). Details of calcium transport in terrestrial isopods has been previously described [START_REF] Ziegler | Microscopical and functional aspects of calcium-transport and deposition in terrestrial isopods[END_REF]. At the same time, the 2 nd stage of vitellogenesis begins for females preparing a parturial moult while the production of vitellogenin and ecdysteroids increases [START_REF] Steel | Ecdysteroid titres in haemolymph and other tissues during moulting and reproduction in the terrestrial isopod, Oniscus asellus (L.)[END_REF][START_REF] Vafopoulou | Vitellogenesis in the terrestrial isopod, Oniscus asellus (L.): Characterization of vitellins and vitellogenins and changes in their synthesis throughout the intermoult cycle[END_REF]. During the stage D 2-4 , the calcium plates are larger and the production of vitellogenin and ecdysteroids is at the maximum [START_REF] Steel | Stages of the intermoult cycle in the terrestrial isopod Oniscus asellus and their relation to biphasic cuticle secretion[END_REF][START_REF] Steel | Ecdysteroid titres in haemolymph and other tissues during moulting and reproduction in the terrestrial isopod, Oniscus asellus (L.)[END_REF][START_REF] Vafopoulou | Vitellogenesis in the terrestrial isopod, Oniscus asellus (L.): Characterization of vitellins and vitellogenins and changes in their synthesis throughout the intermoult cycle[END_REF]. The attractiveness of females varies a lot during their moulting and reproductive cycles [START_REF] Steel | Mechanisms of coordination between moulting and reproduction in terrestrial isopod Crustacea[END_REF]. Their maximum receptivity corresponds to the end of a preparturial or parturial intermoult when the second stage of vitellogenesis occurs [START_REF] Steel | Stages of the intermoult cycle in the terrestrial isopod Oniscus asellus and their relation to biphasic cuticle secretion[END_REF][START_REF] Moreau | The shape of calcium carbonate deposits as an external marker for female reproductive status in terrestrial isopods[END_REF][START_REF] Moreau | Operational sex ratio in terrestrial isopods: interaction between potential rate of reproduction and Wolbachia-induced sex ratio distortion[END_REF]. If abiotic conditions are favorable, females can have two successive clutches during one breeding season either using only stored sperm or by remating after having released the first clutch [START_REF] Verne | Male mating success during parturial intermoults in the terrestrial isopod Armadillidium vulgare revealed by the use of a microsatellite locus[END_REF].

The moulting cycle of A. vulgare, commonly named woodlice or pillbug (Figure S1), is very well described but its impact on chemical communication has not been investigated. A. vulgare excrete an aggregation pheromone and the results from some studies suggest that aggregation might also play a role in reproduction in this species [START_REF] Caubet | Geographical variability of aggregation in terrestrial isopods: What is the actual significance of such behaviour[END_REF][START_REF] Caubet | Biotic triggers of female reproduction in the terrestrial isopod Armadillidium vulgare Latr[END_REF][START_REF] Lefebvre | On the male-effect in the terrestrial crustacean Armadillidium vulgare (Latreille, 1804)[END_REF][START_REF] Lefebvre | Female-extended control over their reproductive investment: the role of early mating interactions on oocyte maturation in the terrestrial crustacean Armadillidium vulgare (Latreille, 1804)[END_REF]. We used A. vulgare as a model species for studying chemical attractiveness between conspecifics depending on their sex and physiological status.

The aims of this study were to investigate sexual communication in A. vulgare and to test (i) if individuals perceive each others at short distance through chemoreception and (ii) if attractiveness of individuals varies depending on their sex and/or moulting status. We expected attractiveness to be higher between individual of the opposite sex but also to change through the moulting cycle.

Materials and Methods

Animal Rearing and Selection

Specimens of Armadillidium vulgare (Latreille, 1804) were derived from individuals collected in Nice, France in 1967. Specimens have been maintained in laboratory conditions ever since (20uC, natural photoperiod of Poitiers, France 46u409N). Each spring since collection, gravid females have been isolated, offspring have been sexed and males and females separated in different boxes before sexual maturity. The breeding has been controlled in order to avoid inbreeding. Individuals are maintained in plastic boxes (26613 cm) on moistened compost and they are fed with slices of fresh carrots and dried leaves of linden (Tilia sp.) ad libitum.

As experience can influence female preference both in vertebrates and invertebrates ( [START_REF] Rutledge | Exposure to multiple sensory cues as a juvenile affects adult female mate preferences in wolf spiders[END_REF] and references therein), we used one-year old virgin individuals of similar size and sexually mature (size .0.7 mm; see [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF]). Females were also sorted according to their physiological status (see fig. 1). They were tested either when in di-ecdysis or at the beginning of pre-ecdysis, before the appearance of the white plates (C/D 0 period), at the appearance of the white plates (middle of pre-ecdysis or D 1 period) or with advanced white plates (end of pre-ecdysis or D 2-4 ). Males were all tested in di-ecdysis or beginning of pre-ecdysis (C/ D 0 ). All tested females were in preparturial intermoult. Their physiological status were confirmed post-experimentally by the shape of the calcium plates [START_REF] Moreau | The shape of calcium carbonate deposits as an external marker for female reproductive status in terrestrial isopods[END_REF].

Choice Chamber

To test the attractiveness of conspecifics in A. vulgare, we used a Y-shaped choice chamber (fig. 2) built in a plastic Petri dish (9.5 cm diameter) covered with a filter paper renewed between each experiment to avoid the presence of retained odours. The plastic elements of the device were cleaned using alcohol everyday. Rigid plastic tunnels were used, with two of them separated in two sections by a mesh in order to create the zones (IIa) and (IIb). This mesh was covered by an opaque paper with tiny holes so that the air could pass through it whilst preventing visual contact between the individuals. These papers were also changed between each experiment. Moreover, two plastic pipettes were sealed at the end of these tunnels to ensure gas exchange with the outside and were used to pulse air regularly (two light pressures at the beginning of the experiment and two others five minutes later) into the system passing through the sections (IIa) and (IIb) to spread the odour of the animals presented for choice.

At the beginning of the experiment, the isopods were in the sections located at the three extreme parts of the tunnels: either one or two target individuals were placed in sections (IIa) and (IIb) (one in each section) and the tested specimen was placed in section (I) and was then able to move into four other sections. The position of target individuals in (IIa) and (IIb) sections were inverted between each experiment so that a preference for the right or left side was not likely to bias the results. Moreover, the target and tested individuals were used only for one test. The target animals were placed in their section 15 minutes before the experiment began so that they got accustomed to the device. The tested individual was then carefully transferred from the group-sorted box to position (I) inside the choice chamber. From the moment the tested specimen entered the neutral section (NS), the time spent in each section and particularly in the left (LS) and the right sections (RS) joining respectively to (IIa) and (IIb) sections was monitored. This was performed by recording the tested individual behaviour using the program EthoLog 2.2 [START_REF] Ottoni | EthoLog 2.2: a tool for the transcription and timing of behavioral observation session[END_REF]. Each test lasted for 10 minutes.

Experimental Groups

Experiments were realized in spring 2009 and 2010, during the natural mating period, always between 2pm and 5pm, in the same conditions of light, temperature and humidity to which animals were raised in (1 Lux, 20uC, 60% 610%).

In the first experiment, the tested individual was placed in a device where a section housed a conspecific while the other was empty (alternatively in section IIa and IIb). In total four combinations were used (Table 1, exp. 1) to test male and female preference between an empty section and a target individual, male or female. Each test was repeated 20 times with different individuals. All the females were in di-ecdysis or at the beginning of pre-ecdysis (C/D 0 period) (Figure 1).

In the second experiment, the tested individual was introduced in a device where 2 target individuals were placed in the isolated sections (IIa and IIb) for each test. We tested the preferences of males and females for conspecifics depending on their sex and physiological status (Table 1, exp.2). We tested male attractiveness between a section with a target male and a target female (either in advanced di-ecdysis, stage C/D 0 or at the end of pre-ecdysis, stage D 2-4 ). Male choice between two females at different moulting stages was also tested with either (i) an early di-ecdysis female C/ D 0 and a pre-ecdysis female D 2-4 with advanced white calcium plates (Figure 1) or (ii) a di-ecdysis female C/D 0 and a pre-ecdysis female D 1 with appearing white calcium plates (alternatively in section IIa and IIb). Choice test with early and advanced di-ecdysis female C/D 0 were defined at the end of the experiment, when females moult. We also tested the choice of females between a target female in the same moulting stage and a target male. This test was performed for both C/D 0 females and D 2-4 females (Figure 1).

In the third experiment, the tested individual had to choose between a chemical extract of one individual (C/D 0 ) placed on inorganic item (a piece of odour-free eraser) fully covered with aluminum and another item only covered by solvent. Chemical extraction was obtained from a total of 20 females. Twenty females were first killed through freezing. Two sets of 10 females were then each submerged in 5 ml of dichloromethane for 24 hours. The remaining solution was then used as chemical extract. The behavioral experiment was repeated 10 times.

Statistical Analysis

The time spent by the tested individual in the right and left sections, was compared using the non-parametric Wilcoxon test for dependent samples. The attractiveness of target individuals was calculated as the median of the time (in seconds) spent by the

Results

We conducted behavioural choice tests in order to compare the attractiveness of conspecifics depending on their sex and physiological status in the terrestrial isopod A. vulgare. Here we present the time that the tested individuals spent in the right and left sections which face the sections housing target conspecifics.

Choice 1: Empty Section Versus Conspecific

Both males and females spent significantly more time in front of the section harbouring a conspecific of the opposite sex (respectively: Wilcoxon: T = 26, N = 20, P = 0.0016 and T = 14, N = 20, P,0.0001; figure 3). In contrast, when presented with a conspecific of the same sex, they did not show any preference for the inhabited section (respectively: Wilcoxon: T = 72, N = 20, P = 0.109 and T = 66, N = 20, P = 0.07; figure 3).

Choice 2: Discrimination between Conspecifics

Male choice. Males spent significantly more time in front of the section housing a female in advanced di-ecdysis (C/D 0 ) than in front of the section with another male (Wilcoxon: T = 36, N = 20, P = 0.005; figure 4). However, this was not observed when the target female was in pre-ecdysis i.e. with advanced white plates and calcium deposit (D 2-4 ) (Wilcoxon: T = 98, N = 20, P = 0.397; figure 4). When the male had to choose between a female without white plates in early di-ecdysis (C/D 0 ) and one in pre-ecdysis (D 2- 4 ), there was no significant preference (Wilcoxon: T = 131, N = 25, P = 0.198; figure 4). However, when the choice test was between a female in di-ecdysis (C/D 0 ) and a female with appearing white plates (D 1 ) the males spent significantly more time in the section close to the female D 1 (Wilcoxon: T = 3, N = 10, P = 0.0063; figure 4).

Female choice. Females in di-ecdysis (C/D 0 ) had a significant preference for the section housing a male rather than for the section housing another female also in di-ecdysis (C/D 0 ) (Wilcoxon: T = 57, N = 20, P = 0.036; figure 5). However, there was no significant preference when the tested female was in preecdysis (D 2-4 ) or when both male and female were in pre-ecdysis (D 2-4 ) (Wilcoxon: T = 97, N = 10, P = 0.382; figure 5).

Choice 3: Male Choice between Female Chemical Extract and Pure Solvent

Males spent significantly more time in front of the section containing an item covered with female extract than in front of the section containing an item covered with solvent only (Wilcoxon: T = 64, N = 10, P = 0.002; figure 6).

Discussion

Aggregation Pheromone and Individual Affinity

Our work revealed an individuals' ability to perceive conspecifics based on olfaction in A. vulgare. Both males and females were significantly more attracted to a conspecific of the opposite sex when the other section was empty. In contrast when they had to choose between a conspecific of the same sex and an empty section there was no significant difference in the time they spent near either sections. Thus we highlight that individual attraction is mainly sex dependent and that the aggregation pheromone is not attractive to all individuals in our experimental design.

Aggregation enhances the emergence of basic cooperation [START_REF] Deneubourg | Dynamics of aggregation and emergence of cooperation[END_REF] and social interactions between conspecifics [START_REF] Costa | The other insect Societies[END_REF]. Aggregation resulting from inter-attractiveness is usually not restricted to individuals of the opposite sex and on the contrary, individuals of the same sex are attracted to each other. In terrestrial isopods, aggregation as a response to pheromones released by conspecifics Table 1. Summary of the different combinations tested and number of replicates (N) for each. has been shown in several species A. vulgare, Oniscus asellus, Porcellio scaber [START_REF] Kuenen | Olfactory orientation in some land-isopods (Oniscoidea, Crustacea)[END_REF]. Using a Y-maze olfactometer, Takeda [START_REF] Takeda | Aggregation phenomenon in terrestrial isopods[END_REF] demonstrated that chemical compounds secreted in the gut and released in faeces constitute an aggregation pheromone for A. vulgare. Since then, most studies on aggregation did not focus on the cues inducing aggregation but on the biotic and abiotic factors influencing its intensity [START_REF] Caubet | Biotic triggers of female reproduction in the terrestrial isopod Armadillidium vulgare Latr[END_REF][START_REF] Hassall | Predicting the effect of climate change on aggregation behaviour in four species of terrestrial isopods[END_REF], some authors even speaking of ''passive aggregation'' due to individual environmental preference for most favorable micro-habitats [START_REF] Smigel | Conglobation in the Pill Bug, Armadillidium vulgare, as a water conservation mechanism[END_REF]. But two recent studies demonstrate that inter-attractiveness between P. scaber conspecifics is stronger than individual preferences which only impacts on the location of aggregates [START_REF] Broly | Aggregation in woodlice: social interaction and density effects[END_REF][START_REF] Devigne | Individual Preferences and Social Interactions Determine the Aggregation of Woodlice[END_REF]; however the authors did not determine the nature of the attractive cues. In another terrestrial isopod species O. asellus, perception of conspecifics seems to be possible by substratum-bound info-chemicals while sexes are mainly distinguished upon antennal contact [START_REF] Stange | What renders a female Oniscus asellus attractive to a soliciting male? A priming approach[END_REF].

Next, we demonstrated that male preference and attraction were significantly higher for the opposite sex. Moreover, males discriminated between females at different moult stages and preferred females at the beginning of pre-ecdysis with appearing white plates (D 1 ) compared to other females at earlier or later stages of their moult cycle. Therefore we suggest that perception cues are air-borne info-chemicals that enable the perception of conspecifics, their sex and even their moulting status in the terrestrial isopod A. vulgare. If vision can be well developed in some Crustaceans and used in communication [START_REF] Marshall | Behavioural evidence for polarisation vision in stomatopods reveals a potential channel for communication[END_REF][START_REF] Marshall | Behavioural evidence for colour vision in stomatopod crustaceans[END_REF], visual acuity seems likely to be extremely reduced in terrestrial forms but could still be used for mate choice. Studies on orientation and relocation of food patches with A. vulgare suggest that both vision and olfaction are used in these processes but this has not been demonstrated yet [START_REF] Tuck | Locating food in a spatially heterogeneous environment: Implications for fitness of the macrodecomposer Armadillidium vulgare (Isopoda: Oniscidea)[END_REF]. In any case, our experimental setup would not allow visual perception of the conspecifics. As for acoustic communication, vibration quality or stridulation (duration and intensity) can be used to discriminate mate quality [START_REF] Conrad | Female choice in the red mason bee, Osmia rufa (L.) (Megachilidae)[END_REF][START_REF] Sivalinghem | Vibratory communication in the jumping spider Phidippus clarus: polyandry, male courtship signals, and mating success[END_REF]. In terrestrial isopods, stridulations appear to be used for defense, as it occurs only when individuals are partially or totally rolled-up and had been only described in Armadillo officinalis [START_REF] Caruso | L'apparato stridulatore e l'emissione di suoni in Armadillo officinalis Dumeril (Crustacea, Isopods, Oniscoidea)[END_REF]. We find it more likely that the attractiveness of the different individuals tested was determined by their odour. In another terrestrial isopod species, O. asellus, chemical cues were shown to signal the presence of conspecifics and to increase male activity in areas previously marked by females [START_REF] Stange | What renders a female Oniscus asellus attractive to a soliciting male? A priming approach[END_REF]. To demonstrate the role of olfaction, we extracted the female odours and applied them on inorganic items. Female odour extract attracted males without other stimulated cues. The next step would be to check whether non-attractive individuals become attractive after coating them with the odour of attractive individuals.

Physiological changes are commonly related with individual odour changes. The attractiveness increase across intermoult phase could be linked with physiological development and odour variation. Numerous changes occur during the second stage of vitellogenesis such as an increasing production of vitellogenin and other proteins [START_REF] Vafopoulou | Vitellogenesis in the terrestrial isopod, Oniscus asellus (L.): Characterization of vitellins and vitellogenins and changes in their synthesis throughout the intermoult cycle[END_REF], the release of ecdysteroids [START_REF] Steel | Ecdysteroid titres in haemolymph and other tissues during moulting and reproduction in the terrestrial isopod, Oniscus asellus (L.)[END_REF], and the translocation of calcium [START_REF] Steel | Storage and translocation of integumentary calcium during the moult cycle of the terrestrial isopod Oniscus asellus (L.)[END_REF], which could induce a change of odours and attractiveness. The production of 20 hydroxyecdysone (20 HD) is linked to ovary development and it functions as a pheromone for some species, but recent evidence shows that this is not as straightforward [START_REF] Chang | The crustacean endocrine system and pleiotropic chemical messengers[END_REF]. For example 20 HD is strongly associated with individual's moult cycle and plays a role in the pheromone involved in reproductive behaviour of the shore crab Carcinus maenas, but does not initiate pair formation [START_REF] Hardege | Identification of crustacean sex pheromone[END_REF].

Aggregation as the First Step to Mate Finding?

The first function of aggregation in Oniscidea is most likely to limit desiccation [START_REF] Smigel | Conglobation in the Pill Bug, Armadillidium vulgare, as a water conservation mechanism[END_REF]. However in species well adapted to terrestrial life, as in A. vulgare, the thick cuticle and conglobation behaviour are already very effective to prevent water loss [START_REF] Smigel | Conglobation in the Pill Bug, Armadillidium vulgare, as a water conservation mechanism[END_REF]. Aggregating behaviour may be conserved for additional benefits which could actually be linked to reproduction. Indeed, aggregation can permit an acceleration of vitellogenesis and the synchronization of moult cycles in females when they are in a group and even more when males are present [START_REF] Caubet | Biotic triggers of female reproduction in the terrestrial isopod Armadillidium vulgare Latr[END_REF][START_REF] Lefebvre | On the male-effect in the terrestrial crustacean Armadillidium vulgare (Latreille, 1804)[END_REF][START_REF] Lefebvre | Female-extended control over their reproductive investment: the role of early mating interactions on oocyte maturation in the terrestrial crustacean Armadillidium vulgare (Latreille, 1804)[END_REF]. Aggregation could reduce the time spent on finding mates and increase mating opportunities. Indeed, we found that even if all conspecifics were attracted to each other, individuals from the opposite sex were the most attractive. As a consequence, inside aggregates, chemical cues could be used to find potential mates.

On the other hand, aggregation might also increase male-male competition. Indeed, in A. vulgare, female receptivity is limited to a very short period while males are sexually active all year round except when moulting [START_REF] Moreau | Operational sex ratio in terrestrial isopods: interaction between potential rate of reproduction and Wolbachia-induced sex ratio distortion[END_REF]. In aquatic species, pre-copulatory mate-guarding is a strategy by which males can monopolize a female mate [START_REF] Henf | Mate-recognition, mate-choice and monopolisation in isopods: an inter-habitat comparison[END_REF][START_REF] Jormalainen | Precopulatory mate guarding in crustaceans: male competitive strategy and intersexual conflict[END_REF]. However, in terrestrial isopods such as A. vulgare, this strategy is not observed. Several explanations have : beginning, middle and end of pre-ecdysis, E: ecdysis; triangle: appearance and development of white calcium plates; the evolution of attractiveness during di-ecdysis is approximated according to the difference that was observed between attractiveness of post-ecdysis and pre-ecdysis females). The hemolymph ecdysteroid titers curve is adapted from Chang and Mykles [START_REF] Chang | Regulation of crustacean molting: A review and our perspectives[END_REF]. The female attractiveness to males curve is based on the current results (plain lines) and unknown attractiveness evolution (dots). doi:10.1371/journal.pone.0057737.g007 been proposed for this difference including higher costs of mateguarding in terrestrial compared to aquatic environment, maleinduced female resistance and capacity of sperm storage by females in terrestrial species [START_REF] Henf | Mate-recognition, mate-choice and monopolisation in isopods: an inter-habitat comparison[END_REF][START_REF] Zimmer | Why do male terrestrial isopods (Isopoda: Oniscidea) not guard females?[END_REF]. Indeed, sperm can be stored for 12 months by females A. vulgare and be used to fertilize another clutch [START_REF] Verne | Male mating success during parturial intermoults in the terrestrial isopod Armadillidium vulgare revealed by the use of a microsatellite locus[END_REF][START_REF] Moreau | Female remating and sperm competition patterns in a terrestrial crustacean[END_REF][START_REF] Suzuki | Structural investigation of the female genitalia and sperm storage sites in the terrestrial isopod Armadillidium vulgare (Crustacea, Isopoda)[END_REF]. Even if the female remates with a second male, at least 50% of the brood will be sired by the first male due to stored sperm precedence. In A. vulgare, mating induces an immediate refractory period during which females reject male mating attempts [START_REF] Moreau | Female remating and sperm competition patterns in a terrestrial crustacean[END_REF]. All these parameters make it very beneficial for a male to locate receptive females before any other male. This is congruent with the hypothesis of Lefebvre et al. [START_REF] Lefebvre | Sexual dimorphism in the antennae of terrestrial isopods: a result of male contests or scramble competition?[END_REF] about antennal sexual dimorphism being a consequence of male scramble competition. Here we show that males can locate receptive females, using short-distance chemical cues. Indeed, we found that females starting the second stage of vitellogenesis (start of pre-ecdysis or D 1 ) were the most attractive, which matches their period of higher receptivity (Figure 7). On the other hand, females at the end of pre-ecdysis (D 2-4 ) were less attractive than other females and they were not particularly more attracted to males than to other females. Eggs are fertilized when laid in the marsupium after the parturial moult, by passing through the oviduct where the sperm is stored [START_REF] Suzuki | Structural investigation of the female genitalia and sperm storage sites in the terrestrial isopod Armadillidium vulgare (Crustacea, Isopoda)[END_REF]. Males could be less attracted to females at this stage because they are more likely to have already mated and stored sperm. These females might as well not be receptive anymore and resist mating attempts because of the refractory period. Moreover, moulting individuals are likely to be more vulnerable (predation, infection) and might not be so attracted by conspecifics compared to the rest of the time when they are gregarious.

Finally we showed that female preference and attraction were higher for the opposite sex but this preference depended on her physiological status. Females in pre-ecdysis (D 2-4 ) did not prefer males to other females at the same moulting stage. Individual behavior changed during the moult cycle and both males and females were attracted by the opposite sex. In A. vulgare sexual selection could be made by both sex and a possible adaptive consequence of female preference has not been studied yet.

We conclude that A. vulgare can perceive and discriminate between male and female conspecifics as well as their physiological status based on short-distance chemical perception only. Aggregation which is at its highest level during the period of reproduction would promote physiological synchronization and even accelerate ovary maturation [START_REF] Caubet | Biotic triggers of female reproduction in the terrestrial isopod Armadillidium vulgare Latr[END_REF] to reach sexual receptivity. Thus, during the mating season, it would favour mate finding/ prospection and choice. The increasing female attractiveness across the moulting cycle might encourage males to remain close to females until they were sexually receptive. Additional behavioural tests coupled with chemical analysis of cuticle compounds will enable the chemical compounds that elicit individual attractiveness and female sexual receptivity to be characterized.

Figure 1 .

 1 Figure 1. Moulting and reproductive cycle of terrestrial isopod females. Vitellogenesis. --: approximate trend in the production of vitellogenin, o: oocytes (small white: before vitellogenesis, medium light grey: first stage of vitellogenesis, big dark grey: second stage of vitellogenesis); Moulting. NM: normal moult, PM: parturial moult, NI: normal intermoult, PPM: preparturial intermoult, PM: parturial moult; A/B: postecdysis, C: di-ecdysis, D 0 , D 1 , D 2-4 : beginning, middle and end of pre-ecdysis, E: ecdysis ''birth'' means the action of female releasing pulli from the brood pouch into the environment. D : as described by Moreau and Rigaud [40]; *: experimental groups in our tests. doi:10.1371/journal.pone.0057737.g001

Figure 2 .

 2 Figure 2. Schematic view of the Y-shaped choice chamber used to test the detection of conspecifics by short-distance chemoreception in Armadillidium vulgare (I: initial position of tested individual, IIa and IIb : position of target individuals; NS: Neutral Section, IS: Intermediate Section, LS: Left Section, RS: Right section) with two plastic pipettes placed at the end of each tunnels and used to pulse air regularly into the system. doi:10.1371/journal.pone.0057737.g002

20 (

 20 C/D 0 : di-ecdysis and beginning of pre-ecdysis (no calcium plates), D 1 : middle of pre-ecdysis (appearance of calcium plates), D 2-4 : end of pre-ecdysis (advanced calcium plates), see fig.1). doi:10.1371/journal.pone.0057737.t001

Figure 3 .Figure 4 .

 34 Figure 3. Time spent by males and females in the right (RS) and left (LS) sections depending on the presence or absence of conspecific in the adjacent section. (F: female, M: male; C/D 0 : di-ecdysis and beginning of pre-ecdysis (no calcium plates), Ø = empty section with no individual, see fig. 1; NS: non-significant p$0.05, ***: p,0.001). doi:10.1371/journal.pone.0057737.g003

Figure 5 .Figure 6 .

 56 Figure 5. Time spent by females at different moulting stages in the right (RS) and left (LS) sections depending of the sex of the conspecific present in the adjacent section. (F: female, M: male; C/D 0 : di-ecdysis and beginning of pre-ecdysis (no calcium plates), D 2-4 : end of pre-ecdysis (advanced calcium plates), see fig. 1; NS: non-significant p$0.05, **: p,0.01). doi:10.1371/journal.pone.0057737.g005

Figure 7 .

 7 Figure 7. Variation of female attractiveness to males throughout a female intermoult preceding a parturial moult. (o: oocytes (small white: before vitellogenesis, medium light grey: first stage of vitellogenesis, big dark grey: second stage of vitellogenesis); A/B: post-ecdysis, C: diecdysis, D 0 , D 1 , D 2-4: beginning, middle and end of pre-ecdysis, E: ecdysis; triangle: appearance and development of white calcium plates; the evolution of attractiveness during di-ecdysis is approximated according to the difference that was observed between attractiveness of post-ecdysis and pre-ecdysis females). The hemolymph ecdysteroid titers curve is adapted from Chang and Mykles[START_REF] Chang | Regulation of crustacean molting: A review and our perspectives[END_REF]. The female attractiveness to males curve is based on the current results (plain lines) and unknown attractiveness evolution (dots). doi:10.1371/journal.pone.0057737.g007

PLOS ONE | www.plosone.org

March 2013 | Volume 8 | Issue 3 | e57737

Acknowledgments

The figure 1 is modified from Yves Caubet. Figure 1, 2 & 6 were designed by Fanny Beauche ´and Gilles Bosquet using In-design. We thank Joanne Bertaux for her advices and for English revisions on the manuscript and Yves Caubet, Pierre Gre `ve and Catherine Souty-Grosset for their comments and Lucy Ovens for linguistic corrections. We also thank Sylvain Mercier, Emilie Meunier and Lauriane Olivier who contributed to behavioral observations. Finally, we are grateful to Alexandra Laffite who is in charge of terrestrial isopods management and welfare in the Laboratory Ecology Evolution and Symbiosis. Credit for the picture of A. vulgare to Freddie-Jeanne Richard.

Supporting Information


J.-M Nicolas

E Trouvé

R Fallourd

F Vernier

F Tupin

O Harant

M Gay

L Moreau

A FIRST COMPARISON OF COSMO-SKYMED AND TERRASAR-X DATA OVER CHAMONIX MONT-BLANC TEST-SITE

Keywords: SAR images time series, Cosmo-SkyMed, TerraSAR-X, interferometry, correlation, glaciers

This paper presents the first results obtained with satellite image time series (SITS) acquired by Cosmo-SkyMed (CSK) over the Chamonix Mont-Blanc test-site. A CSK SITS made of 39 images is merged with a TerraSAR-X SITS made of 26 images by using the orbital information and co-registration tools developed in the EFIDIR project. The results are illustrated by the computation of speckle-free images by temporal averaging, by the generation and comparison of topographic interferograms and by the measure of glacier displacement fields by amplitude correlation.

INTRODUCTION

Chamonix Mont-Blanc has become one of the experimental test site to demonstrate the potential of Earth Observation data for Alpine glacier monitoring. Various sources of information have been used and merged to measure the evolution of the glacier surface (elevation, displacement fields, classifications...): spaceborne optical images (SPOT) [START_REF] Berthier | Mountain glaciers surface motion derived from satellite optical imagery[END_REF], airborne photography and the first generation of SAR spaceborne data (ERS) [START_REF] Trouvé | Combining airborne photographs and spaceborne SAR data to monitor temperate glaciers. Potentials and limits[END_REF], airborne SAR data acquired by the DLR in 2006 and 2007 (E-SAR) [START_REF] Landes | Monitoring temperate glaciers by high resolution Pol-InSAR data: first analysis of Argentière E-SAR acquisitions and in-situ measurements[END_REF]... This test site has also been proposed by the EFIDIR project teams (http://www.efidir.fr) in response to the Announcements of Opportunities issued from the different space agencies for the launch of the new generation SAR satellites. The EFIDIR project objective is to develop an open platform of archival storage and of processing, fitted on one hand to SAR data specificity and, on the other hand, to large time series of SAR images from which ground motion measures are extracted. The first results obtained with High Resolution data (TerraSAR-X) [START_REF] Fallourd | Monitoring Temperate Glacier Displacement by Multi-Temporal TerraSAR-X Images and Continuous GPS Measurements[END_REF] or fully * jean-marie.nicolas@telecom-paristech.fr polarimetric data (Radarsat-2) [START_REF] Harant | Displacement estimation by maximum likelihood texture tracking[END_REF] show an increased potential in terms of measurement of displacement fields and analysis of the temporal evolutions [START_REF] Julea | PolSAR Radarsat-2 satellite image time series mining over the Chamonix Mont-Blanc test site[END_REF].

In this paper, we present the first results obtained on this test site with Cosmo-SkyMed (CSK) stripmap data (project 2145, "Monitoring glacier activity by combined interferometry and Single Look Complex image processing"). A set of 39 images has been acquired in ascending passes between March 2009 and December 2011. The aim of this first analysis is to provide some comparisons with a set of 26 TerraSAR-X (TSX) stripmap data acquired in ascending passes between May 2009 and September 2011, with a slightly different incidence angle: 34 • for CSK and 44 • for TSX. Despite this difference and its geometrical consequences, we illustrate how such satellite image time series (SITS) can be combined to increase the repetitivity of space-borne SAR observations for the monitoring of fast changing geophysical features such as Alpine glaciers. We first describe the co-registration technique developed to built the time series by using only the orbital information and how it can be used for either TSX or CSK data stacking, and even for direct inter-sensor coregistration to obtain a stack of 65 images mixing CSK and TSX data. We then illustrate the potential of these time series with early results such as speckle-free images, interferometric fringes in coherent areas and displacement measurement by amplitude correlation over glaciers.

CO-REGISTRATION OF THE CSK-TSX DATA SET

Co-registration of SAR images from repeated orbits is a well known task with some specific tools often developed for interferometric applications which requires sub-pixel accuracy. Those developed for the first generation of SAR satellites are usually based on similarity measures to derive geometrical transforms necessary to re-sample the slave image over the master one, and then "substract" the orbital and topographi-cal fringes in a second stage. The increased accuracy of the state vectors provided with the new generation SAR satellites such as TSX and CSK and their higher resolution allows different approaches to be experimented.

In this paper, we perform this resampling by using directly the meta information provided in the TSX xml files or CSK H5 files, without using similarity measurements which are dependant on the image content (textured or homogeneous areas), and very sensitive to surface changes (snow fall, seasonal variations...) and variations of the incidence angles in the case of images acquired from different tracks or from two different sensors such as CSK and TSX. Moreover, by using the pure geometric information associated to the radar principle and the SAR systems (antenna positions, range and azimuth sampling...), we can avoid mixing the offset due to the baseline with the sought-after information: the offset due to the ground surface displacement.

Then, depending on the topography of the scene and on the availability of an accurate DEM, we can implement this approach with or without using a DEM. Without DEM (as illustrated in the rest of this paper), only a constant "mean" altitude is introduced. The results presented in the following section show that this can be sufficient for a local scale observation as it is often the case for ground motion due to land instability, urban subsidence or mountain glaciers. This makes the processing rather straightforward and avoid the requirement of a DEM which can also be a source of errors. In the case of large scale observation, as for seismic deformations, the DEM information can be introduced by computing for each ground points the difference of the distance to the antenna, and projecting this information on the Master image grid as proposed in radar-coding tools [START_REF] Pétillot | Radar-Coding and Geocoding Lookup Tables for the Fusion of GIS and Sar Data in Mountain Areas[END_REF].

EXPERIMENTAL RESULTS

CSK and TSX Chamonix Mont-Blanc data set

The co-registration technique presented in the previous section has been applied to a large data set made of 65 X-band SAR images acquired in ascending passes over Chamonix Mont-Blanc test-site, with 39 Cosmo-SkyMed images and 26 TerraSAR-X images. This technique initially devoted to TSX images has been used to register CSK data without any adaptation (except a slight remaining translation between the two sensors). The main characteristics of the CSK and TSX time series are recalled in Table 1.

This data set covers several well known glaciers of the Mont-Blanc massif, the city of Chamonix and several ski stations of the valley. The Argentière glacier is one of the most studied glaciers in the Chamonix Mont-Blanc valley because of the long history of scientific experimentations and measurements performed on its surface and under the glacier thanks to galleries built to collect water for hydro-electricity.

Figure 1

Temporal averaging

To illustrate the performance of the direct co-registration technique and the potential of the resulting CSK and TSX stacks, we first propose to derive speckle-free images by computing the temporal geometrical mean. The two time series are co-registered independently, then for each stack, the magnitudes are averaged along the temporal axis with a geometrical mean which seems to be pertinent in this temporal framework [8].

The results obtained with the CSK and the TSX time series are illustrated in Figure 2 on the parking area of the "Grand-Montet" cable car. The results can be considered as speckle-free images obtained without losing spatial resolution. Thanks to this averaging, several features become Fig. 2. Speckle removal obtained by temporal averaging of 39 CSK images and 26 TSX images acquired over Chamonix Mont-Blanc. 382x382 pixels sub-image of the "Grand-Montet" cable car parking area. clearly visible like the curves of the parking separations, the aligned strong scattered of skiing facilities...

Interferograms

Although these data have not been acquired in interferometric mode, it is possible to generate interferograms from the coregistered SLC images. The results obtained with a CSK pair and a TSX pair acquired in September 2009 are illustrated in Figure 3. In both cases, the coherence is lost over the glacier due to important surface changes within 16 days (CSK) or 11 days (TSX). On the lateral moraines, the coherence is strong enough to preserve the phase information, with a lower level in the CSK interferograms mostly due to the larger temporal and spatial baselines.

Since the orbital fringes have been removed in the coregistration step, the phase rotation observed in Figure 3 corresponds to topographic fringes and is proportional to the perpendicular baseline B ⊥ . For these two interferograms, the ratio of the perpendicular baselines is equal to 3, which allows a direct comparison of the fringe patterns as illustrated in Fig- ure 4. For each TSX fringe displayed in gray levels, one can observe three CSK fringes displayed in color.

A first test of displacement measurement

In the EFIDIR project, a method based on the Zero-mean Normalized Cross Correlation (ZNCC) allows glacier displacement to be estimated on cracked glaciers [START_REF] Fallourd | Monitoring Temperate Glacier Displacement by Multi-Temporal TerraSAR-X Images and Continuous GPS Measurements[END_REF]. This method has been applied on the co-registered CSK -TSX SITS on the CSK: 2009/09/04-20, B ⊥ =399m TSX: 2009/09/18-29, B ⊥ =133m Fig. 3. Amplitude, phase and coherence over a part of Argentière glacier after time series co-registration (1024x1024 pixels area, 3x3 averaging) cracked area illustrated in Figure 3. The results presented in Figure 5-a) illustrate the ZNCC level obtained for the best matching with 51x51 pixels windows. The range and azimuth offsets are converted in meter/day and illustrated together with the 2D displacement magnitude in Figure 5-b)-c) and d) respectively. The glacier displacement in the North direction is clearly observed whereas the LOS displacement and the azimuth displacement on motion-free areas are close to zero. Both results are consistent, with less noise (wrong matching) with the TSX data which are more correlated.

CONCLUSIONS

After this positive experiment on combining CSK and TSX time series, the EFIDIR teams aim to develop automatic tools to analyse long SAR SITS. On the Chamonix Mont-Blanc glaciers, further research includes the determination of displacement charts with long time series of CSK data corresponding to 2010 and 2011 summers combined with TerraSAR-X time series from 2009 to 2011. By this way, a global analysis of these glaciers during several years can be provided to glaciologists to complement spatially and temporally the in-situ measurements.

Fig. 1 .

 1 Fig. 1. CSK and TSX acquisitions over Argentière and Merde-Glace glaciers on May 5, 2011 in their initial geometry

Fig. 4 .

 4 Fig. 4. Composition of the TSX (in gray) and CSK (in color) topographic fringes, on a 540x640-pixels area (lower left corner of Figure 3.

Table 1 .

 1 CSK and TSX data set acquired in ascending passes

	CSK	TSX

illustrates the Argentière and Mer-de-Glace over Chamonix Mont-Blanc test-site glaciers visibility on CSK and TSX data. Even if the CSK local incidence angle is lower than with TSX, creating more noticeable foldover artefacts, it is possible to analyse the totality of Argentière glacier with this CSK and TSX dataset.

CSK (4560x6360 pix.) TSX (5120x5850 pix.)

[8] G. Quin, B. Pinel-Puyssegur, and J.-M. Nicolas. Comparison of harmonic, geometric and arithmetic means for change detection in SAR time series. In EUSAR 2012 -9th European Conference on Synthetic Aperture Radar, Nuremberg, Germany, April 2012.

ACKNOWLEDGMENTS

This work was supported by the EFIDIR project(ANR-07-MDCO-004, http://www.efidir.fr), granted by the French National Research Agency (ANR). The authors wish to thank the German Space Agency (DLR) for providing the TerraSAR-X data (Project MTH0232) and the Italian Space Agency (ASI) for providing the CSK data (Project 2145).