Anthony G E Mathiron
email: anthony.mathiron@sfr.fr

Ryan L Earley

Marlène Goubault

Juvenile hormone manipulation affects female reproductive status and aggressiveness in a non-social parasitoid wasp

Keywords: juvenile hormone, reproductive maturation, aggressive behaviors, conflict resolution

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

Introduction

As gaining access to resources brings fitness benefits to individuals, both vertebrate and invertebrate animals commonly exhibit aggressive behaviors during conflicts over such resources [START_REF] Collias | Aggressive behavior among vertebrate animals[END_REF][START_REF] Breed | Hormonal influences on invertebrate aggressive behavior[END_REF][START_REF] Briffa | Analysis of animal contest data[END_REF]. For example, in parasitoid insects, hosts represent an indivisible resource over which females compete to lay their eggs, and physical interactions between females for host access have been observed in various parasitoid species [START_REF] Hughes | Host-searching behavior of Venturia canescens (Grav.) (Hymenoptera: Ichneumonidae): Interference-the effect of mature egg load and prior behavior[END_REF][START_REF] Petersen | The importance of being larger: parasitoid intruder -owner contests and their implications for clutch size[END_REF][START_REF] Field | Don't count your eggs before they're parasitized: contest resolution and the trade-offs during patch defense in a parasitoid wasp[END_REF][START_REF] Goubault | Does host value influence female aggressiveness, contest outcome and fitness gain in parasitoids?[END_REF][START_REF] Mohamad | Can subjective resource value affect aggressiveness and contest outcome in parasitoid wasps?[END_REF][START_REF] Mathiron | Let the most motivated win : resource value components affect contest outcome in a parasitoid wasp[END_REF]. In many animal taxa, aggressive conflicts have also been observed between individuals for sexual partners [START_REF] West-Eberhard | Sexual Selection, Social Competition, and Evolution[END_REF][START_REF] Schwagmeyer | Mating Competition in an Asocial Ground-Squirrel, Spermophilus tridecemlineatus[END_REF][START_REF] Simmons | Intermale Competition and Mating Success in the Field Cricket, Gryllus bimaculatus (Degeer)[END_REF], food [START_REF] Barton | Sociospatial mechanisms of feeding competition in female olive baboons, Papio anubis[END_REF][START_REF] Vogel | Rank differences in energy intake rates in white-faced capuchin monkeys, Cebus capucinus: The effects of contest competition[END_REF], territories [START_REF] Rowland | The effects of body size, aggression and nuptial coloration on competition for territories in male threespine sticklebacks, Gasterosteus aculeatus[END_REF][START_REF] Festa-Bianchet | Aggression among Lekking Male Fallow Deer (Dama dama): Territory Effects and Relationship with Copulatory Success[END_REF], and dominance status [START_REF] Chase | Models of hierarchy formation in animals societies[END_REF]. However, being constantly aggressive can be costly, as potential physical injuries can lead to death [START_REF] Palombit | Lethal territorial aggression in a white-handed gibbon[END_REF][START_REF] Innocent | Lethal combat over limited resources: Testing the importance of competitors and kin[END_REF], and decreased vigilance can increase predation risk [START_REF] Jakobsson | Escalated fighting behaviour incurs increased predation risk[END_REF][START_REF] Díaz-Uriarte | Anti-predator behaviour changes following an aggressive encounter in the lizard Tropidurus hispidus[END_REF]. Animals should therefore evaluate the costs and benefits of exhibiting agonistic behaviors and then behave appropriately [START_REF] Maynard-Smith | The logic of animal conflict[END_REF].

Endocrine regulation is one important way that animals can match behavioral and physiological responses to their current environment [START_REF] Tibbetts | The challenge hypothesis across taxa: Social modulation of hormone titres in vertebrates and insects[END_REF]. In vertebrates, testosterone is generally involved in the modulation of aggressive behaviors during conflicts associated with reproduction, such as the establishment and maintenance of breeding territories, mate access, and offspring defense [START_REF] Wingfield | Androgens and Mating Systems: Testosterone-Induced Polygyny in Normally Monogamous Birds[END_REF]. For example, [START_REF] Salvador | The cost of producing a sexual signal: Testosterone increases the susceptibility of male lizards to ectoparasitic infestation[END_REF] showed that testosterone supplementation in large males of the territorial lizard Piammodromu algirus increased the number of chasing behaviors toward conspecific intruders. Moreover, during mate choice, female zebra finches treated with testosterone were more aggressive toward conspecific females than control individuals (Adkins-Regan, 1999). Whilst well-described in vertebrates, the endocrine basis of behavior is still relatively unknown in insects [START_REF] Zera | Endocrine analysis in evolutionary-developmental studies of insect polymorphism: Hormone manipulation versus direct measurement of hormonal regulators[END_REF][START_REF] Tibbetts | The challenge hypothesis across taxa: Social modulation of hormone titres in vertebrates and insects[END_REF]. Because vertebrates and insects have similar behaviors but different endocrine systems [START_REF] Nijhout | Insect Hormones[END_REF][START_REF] Adkins-Regan | Hormones and Animal Social Behavior[END_REF], exploring how hormones modulate insects behaviors can therefore provide insight into the evolution of endocrine regulation of behavior [START_REF] Emlen | The Development and Evolution of Exaggerated Morphologies in Insects[END_REF].

In insects, juvenile hormone (JH) is a good candidate for studying the flexibility of endocrine responses to intraspecific conflict because it has important effects on both reproductive processes and behavior. Together with nutrition (see the review of [START_REF] Wheeler | The Role of Nourishment in Oogenesis[END_REF], JH plays an important and diverse role in reproduction in many adult insect species by regulating vitellogenesis in the fat body and vitellogenin uptake by the ovaries (reviewed by [START_REF] Tobe | Structure and Regulation of the Corpus Allatum[END_REF][START_REF] Nijhout | Insect Hormones[END_REF][START_REF] Robinson | Juvenile hormone in adult eusocial hymenoptera: Gonadotropin and behavioral pacemaker[END_REF][START_REF] Hartfelder | Insect juvenile hormone: from "status quo" to high society[END_REF]. In Hymenoptera, JH is involved in the regulation of ovarian development in the facultatively eusocial sweat bee Megalopta genalis [START_REF] Smith | Juvenile hormone levels reflect social opportunities in the facultatively eusocial sweat bee Megalopta genalis (Hymenoptera : Halictidae)[END_REF] and in primitively social species like polistine wasps [START_REF] Barth | Juvenile hormone promotes dominance behavior and ovarian development in social wasps (Polistes annularis)[END_REF], halictine bees [START_REF] Bell | Factors controlling initiation of vitellogenesis in a primitively social bee, Lasioglossum zephyrum (Hymenoptera: Halictidae)[END_REF] and bumblebees [START_REF] Bloch | Juvenile hormone titers, juvenile hormone biosynthesis, ovarian development and social environment in Bombus terrestris[END_REF][START_REF] Amsalem | Precocene-I inhibits juvenile hormone biosynthesis, ovarian activation, aggression and alters sterility signal production in bumble bee (Bombus terrestris) workers[END_REF]. However, in the highly social honeybee Apis mellifera, JH is apparently involved in the regulation of division of labor but has no clear role in the 4/24 regulation of reproduction (reviewed in [START_REF] Robinson | Regulation of Division of Labor in Insect Societies[END_REF][START_REF] Robinson | Juvenile hormone in adult eusocial hymenoptera: Gonadotropin and behavioral pacemaker[END_REF]. In many eusocial hymenopterans, JH is also associated with displays of behaviors often judged to be aggressive [START_REF] Tibbetts | The challenge hypothesis across taxa: Social modulation of hormone titres in vertebrates and insects[END_REF]. For example, in the epiponine wasp Polybia micans, JH may fuel aggressive behaviors of single queens heading a colony (Kelstrup et al. 2014a). Similar observations have been made in paper wasps, in which high JH titers are associated with dominance behaviors [START_REF] Röseler | The Activity of Corpora Allata in Dominant and Subordinated Females of the Wasp Polistes Gallicus[END_REF][START_REF] Röseler | Influence of insect hormones on the establishment of dominance hierarchies among foundresses of the paper wasp, Polistes gallicus[END_REF][START_REF] Giray | Juvenile hormone, reproduction, and worker behavior in the neotropical social wasp Polistes canadensis[END_REF][START_REF] Tibbetts | The Challenge Hypothesis in an Insect: Juvenile Hormone Increases during Reproductive Conflict following Queen Loss in Polistes Wasps[END_REF][START_REF] Tibbetts | Juvenile hormone influences precontest assessment behaviour in Polistes 24/24 dominulus paper wasps[END_REF]Kelstrup et al. 2016; but see [START_REF] Kelstrup | Polistes smithii vs. Polistes dominula: the contrasting endocrinology and epicuticular signaling of sympatric paper wasps in the field[END_REF]. In honeybees, JH titers are higher in more aggressive workers and JH treatments cause honeybees to exhibit more frequent guarding behaviors [START_REF] Sasagawa | Hormonal Control of the Division of Labor in Adult Honeybees (Apis mellijlera L.) I. Effect of Methoprene on Corpora Allata and Hypopharyngeal Gland, and Its cr-Glucosidase Activity[END_REF][START_REF] Pearce | Juvenile hormone and aggression in honey bees[END_REF]. However, JH has no effect on aggression in the swarmfounding epiponine wasp Synoeca surinama (Kelstrup et al., 2014b) and may even be negatively correlated with aggression in the queenless ponerine ant, Streblognathus peetersi [START_REF] Brent | Hormonal correlates of reproductive status in the queenless ponerine ant, Streblognathus peetersi[END_REF]. Surprisingly, no work has yet focused on the relationship between JH, reproduction, and aggressiveness in strict non-social Hymenoptera species, although it may help to understand how JH actions have evolved across insect taxa with different degrees of sociality.

In the present study, we investigated how JH treatment influences: i) female reproductive status, and ii) aggressive behaviors exhibited during contests over host access as well as the outcome of such contests, using Eupelmus vuilleti Crawford (Hymenoptera: Eupelmidae). Eupelmus vuilleti is a solitary ectoparasitoid (i.e. only one juvenile can develop per host, with supernumerary offspring being killed during larval competition). Females parasitize and feed upon larvae and pupae of Callosobruchus maculatus (Fab) (Coleoptera: Bruchidae), which infest cowpea seeds, Vigna unguiculata (L.) Walp (Fabaceae; [START_REF] Terrasse | Distribution de la ponte et évitement du superparasitisme chez l'hyménoptère solitaire Bruchocida vuilleti Cwf. (Hym., Eupelmidae), parasite des stades larvaires de son hôte, Callosobruchus maculatus F. (Col., Bruchidae)[END_REF]. When several females are simultaneously present on a host patch, they tend to protect the host that they exploit by displaying aggressive behaviors towards conspecific competitors [START_REF] Mohamad | Can subjective resource value affect aggressiveness and contest outcome in parasitoid wasps?[END_REF][START_REF] Mathiron | Let the most motivated win : resource value components affect contest outcome in a parasitoid wasp[END_REF].

Previous studies have investigated the role of different factors (e.g., egg load, host density and host quality) on aggressiveness and contest outcomes in E. vuilleti [START_REF] Mohamad | Can subjective resource value affect aggressiveness and contest outcome in parasitoid wasps?[END_REF][START_REF] Mohamad | Wait or fight? Ownership asymmetry affects contest behaviors in a parasitoid wasp[END_REF][START_REF] Mohamad | Do resource value and ownership status affect intensity and resolution of contests in a parasitoid wasp?[END_REF] competition for host access.

While measuring variation in hormone titers is an important first step to understand the endocrine basis of phenotypic plasticity [START_REF] Zera | Endocrine analysis in evolutionary-developmental studies of insect polymorphism: Hormone manipulation versus direct measurement of hormonal regulators[END_REF], one method that allows for understanding individual responses to hormonal change is to administer a similar amount of hormone to numerous individuals and, subsequently, to test how hormone manipulations affect the phenotype [START_REF] Tibbetts | Hormones and Behavior Endocrine mediated phenotypic plasticity : Conditiondependent effects of juvenile hormone on dominance and fertility of wasp queens[END_REF]. As such, manipulation of JH is one of the most common methods employed to understand its mode of action and functions in insects [START_REF] Sláma | Insect juvenile hormone analogues[END_REF][START_REF] Ramaseshadri | Recent Progress in Juvenile Hormone Analogs[END_REF]. A first experiment was therefore designed to determine if JH treatment affected female reproductive status. As for many other insect species, we expected JH to have a gonadotropic effect in females, i.e. that wasps treated with JH would have more ready-to-lay eggs than control females (i.e. treated with vehicle). In a second experiment, we studied whether aggressiveness and resolution of conflicts between females were influenced by a JH treatment. We expected that individuals treated with JH would display more aggressive behaviors and would ultimately be more likely to win contests for hosts relative to control females.

Material and Methods

Laboratory breeding

In 2007, E. vuilleti emerged from cowpea seeds collected in Togo (West Africa) and infested by the bruchid beetle Callosobruchus maculatus. Parasitoids were then bred in the laboratory (IRBI, University of Tours, France) on larval and pupal stages of C. maculatus, according to the methodology described by [START_REF] Jaloux | Interspecific relationships between the solitary ectoparasitoid, Eupelmus vuilleti (Crw.) (Eupelmidae), and its sympatric species, Dinarmus basalis (Rond.) (Pteromalidae), in the presence of their host, Callosobruchus maculatus pic (Coleoptera Bruchidae)[END_REF]. All experimental procedures were carried out in a climate room at 30°C and 12:12 h light: dark. Cowpea seeds were dissected to collect hosts for E. vuilleti, which were then individually placed in a standard transparent gelatin capsule (length: 2 cm, diameter: 0.6 cm). This system mimics a cowpea seed without significantly altering the oviposition 6/24 behavior of females, and facilitates observation of the number of eggs laid by females [START_REF] Gauthier | Could the egg itself be the source of the oviposition deterrent marker in the ectoparasitoid Dinarmus basalis?[END_REF]. Previous studies have shown that after a period of acclimation, parasitoid females behave in the same manner to the presence of hosts inside seeds and capsules [START_REF] Gauthier | Could the egg itself be the source of the oviposition deterrent marker in the ectoparasitoid Dinarmus basalis?[END_REF][START_REF] Jaloux | Interspecific relationships between the solitary ectoparasitoid, Eupelmus vuilleti (Crw.) (Eupelmidae), and its sympatric species, Dinarmus basalis (Rond.) (Pteromalidae), in the presence of their host, Callosobruchus maculatus pic (Coleoptera Bruchidae)[END_REF], although the number of eggs laid on hosts is slightly lower inside capsules [START_REF] Damiens | Egg-laying, pre-imaginal growth dynamics, and mortality in Eupelmus orientalis and Dinarmus basalis, two solitary ectoparasitoids of Callosobruchus maculatus[END_REF].

Females used in the following experiments emerged in isolation, which prevented them from having any previous experience of oviposition or competition. We placed them individually in petri dishes (diameter: 8.5 cm; height: 2.7 cm), and provided cotton soaked in water and one pupa of C. maculatus located in gelatin capsule that was renewed each day preceding the experiments. E. vuilleti is a synovigenic parasitoid species: emerging females possess a few mature eggs and they mature additional eggs throughout their adult life. Oviposition activity increases over time and peaks three days after emergence [START_REF] Jaloux | Interspecific relationships between the solitary ectoparasitoid, Eupelmus vuilleti (Crw.) (Eupelmidae), and its sympatric species, Dinarmus basalis (Rond.) (Pteromalidae), in the presence of their host, Callosobruchus maculatus pic (Coleoptera Bruchidae)[END_REF]. To stimulate oogenesis [START_REF] Terrasse | Distribution de la ponte et évitement du superparasitisme chez l'hyménoptère solitaire Bruchocida vuilleti Cwf. (Hym., Eupelmidae), parasite des stades larvaires de son hôte, Callosobruchus maculatus F. (Col., Bruchidae)[END_REF], we also gave one male to females during the first day.

Experiment 1: Effect of JH on females' reproductive status

To investigate the influence of JH treatment on female reproductive status, two-day old wasps were randomly assigned to one of the five following treatments: they received either 0.04 µL of acetone (N=36) or 0.04 µL of JHIII (purchased from Sigma-Aldrich, Inc.) dissolved in acetone at four different concentrations (8, 16, 32 or 64 pg/µl; N=35, N=39, N=39, N=37, respectively). The final volume was established based on preliminary tests showing that mean haemolymph volume in E. vuilleti females is 0.175 µl (but this volume may vary depending on body mass, physiological state or environmental conditions; Borde et al., unpublished data). Immediately after being immobilized on a petri dish that was cooled by ice, wasps were injected into the abdomen through the intersegmental membrane using the nanoliter injector Nanoject III (Drummond Scientific Company). Females were then individually placed in a petri dish and were frozen at -20°C either 4h (N=20 for injections of 0, 8, 16, 32 pg/µl and N=18 for injection of 64 pg/µl) or 24h (N=16 for after injection. Females were deprived of hosts so that no mature eggs were laid during this period.

Finally, all females were weighed using an electronic balance (Ohaus Discovery® model, accuracy: 0.01mg) and then dissected to count mature (i.e. ready-to-lay) oocytes in their abdomen.

Experiment 2: Effect of JH on females' aggressiveness and contests outcome

To investigate the influence of JH manipulation on female aggressive behavior, three-days old wasps were randomly assigned to two different injection treatments: females injected with either 0.04 µL of JHIII diluted in acetone (concentration = 32 pg/µl) or 0.04 µL of acetone alone. Wasps were marked on the dorsal part of their thorax with a dot of bright yellow or bright red acrylic paint the day before behavioral observations to allow individual identification during contests. Paint color did not affect contest outcomes (wasps painted red won 17 of 25 contests; binomial test: P=0.11).

Females were deprived of hosts for at least 2h before the start of the contest so that they were more ready to oviposit. At the beginning of the observation, two wasps were simultaneously introduced into an apparatus consisting of a plastic block made of three chambers linked by a narrow channel [START_REF] Petersen | The importance of being larger: parasitoid intruder -owner contests and their implications for clutch size[END_REF]Fig. 1.). All contests were run between one control female (i.e., injected with acetone only) and one test female (i.e., injected with JHIII), challenging for a 4 th instar larva of C. maculatus located in gelatin capsule and previously placed in the central chamber of the contest arena (Fig. 1.). [START_REF] Mathiron | Let the most motivated win : resource value components affect contest outcome in a parasitoid wasp[END_REF] showed that females with previous oviposition experience on pupae were more motivated to gain access to a 4 th instar larva. Contests occurred between females of the same age (3 days old), marked with different colors and visually matched for size; post-experiment data analysis confirmed that contestants of the same dyad did not differ significantly in body mass (Wilcoxon signed-ranks test: T=147.5, N=25, P=0.7). Both females could freely move throughout the entire contest arena. Observations lasted 1h or were stopped when: i) one female pushed her opponent out of the central chamber, ii) one female exited the central chamber for at least 2 min, or iii) neither female touched the host nor displayed aggressive interactions for at least 2 min.

8/24

During all tests, we recorded oviposition and aggressive behaviors displayed by each female.

Upon detection of a conspecific (females raised their antennae in the direction of their opponent), females frequently interrupted their behavioral oviposition sequence [START_REF] Mohamad | Can subjective resource value affect aggressiveness and contest outcome in parasitoid wasps?[END_REF]. They either simply kick their opponent with their legs without taking their ovipositor out of the capsule (defensive behavior) or can escalate to a full attack, in which case one female generally chases her opponent away from the capsule, hits her with her head or mounts her [START_REF] Mathiron | Let the most motivated win : resource value components affect contest outcome in a parasitoid wasp[END_REF].

Chasing, hitting and mounting can lead to the loser leaving the central chamber of the arena (8/25 loser females left the central chamber after an opponent attack during this experiment). Only these behaviors, collectively termed as 'attacks', were therefore considered in the rest of the study. When both females of a dyad stayed in the central chamber during the whole time of observation, the winning female was determined as the one exhibiting oviposition behaviors.

Wasps were immediately frozen at -20°C after the experiments, weighed using an electronic balance (Ohaus Discovery® model, accuracy: 0.01mg), and dissected to count the number of mature and non-mature eggs in their abdomen. Finally, after each contest, we counted the number of eggs laid on the host by the winning female to determine their mature egg load before the contests (i.e., initial mature egg load).

Statistical analysis

We performed data analyses with the software Rstudio (1.0.143 version -© 2009Rstudio (1.0.143 version -© -2016 RStudio, Inc.), using α=0.05. Our general approach was to use, when possible, parametric analyses in which the assumed distribution of residuals was matched to the data rather than transforming data to fit standard assumptions [START_REF] Wilson | Statistical analysis of sex ratios: an introduction[END_REF][START_REF] Briffa | Analysis of animal contest data[END_REF]. However, we used nonparametric tests when parametric conditions were not verified.

Experiment 1: We first investigated whether the mature egg load of females differed significantly between JHIII treatments, 4h after injection, by performing a general linear model (GLM) assuming a Poisson distribution of errors. We then ran a linear model (LM) and a polynomial 10/24 linear model (PLM) to explore the effect of JHIII treatments on the number of mature eggs 24h after injection, and we compared the linear and polynomial models to determine which one provided a better fit.

Experiment 2: We first verified that initial mature egg load did not differ between JHIII females (mean egg load ± S.E.: 6.2 ± 0.7) and control females (mean egg load ± S.E.: 5 ± 0.5; Student test: t25=-1.41, P=0.16). We then performed a generalized linear mixed model (GLMM) with a Poisson error distribution to explore the influence of contestants' initial mature egg load and injection treatment on the number of attacks (calculated as the sum of chasing, hitting, and mounting behaviors) displayed by females, with contest identity as a random factor. Lastly, we conducted a GLMM assuming a binomial error distribution to explore the influence of initial mature egg load of females and injection treatments on contest outcomes, with contest identity as a random factor. To run this analysis, we defined contest outcome as a binary response: 0 = the female lost and 1 = the female won.

Results

Experiment 1: Effect of JH on female reproductive status

The number of mature eggs in females did not vary across treatments 4h after injections (GLM: χ 2 =0.006, df=1, P=0.94; Fig. 2a). However, 24h after injections, mature egg load was influenced significantly by treatment (LM: F1,86=4.05, P=0.047, R²=0.335).

Moreover, we found that the polynomial model fitted better than the linear model, with a dose-dependent effect of JHIII injection on egg maturation reaching a plateau at 64 pg/µL (PLM: F1,85=4.05, P=0.047, R²=0.365; Fig. 2b).

Experiment 2: Effect of JH on female aggressiveness and contests outcome

The number of attacks displayed by females was influenced significantly by the interaction between female egg load and injection treatment (Table 1). Wasps attacked significantly more during a contest when they had more ready-to-lay eggs, and this effect was stronger when females were injected with JHIII compared to control females injected with acetone alone (Table 1, Fig. 3). Effect of female egg load x injection treatment interaction 12/24 was still significant when removing data from the contest with a JHIII female displaying 22 attacks and her control opponent displaying 2 attacks (GLMM: χ²=4.87, df=1, P=0.03). The probability of a female winning a contest was not influenced significantly by either the interaction between her initial mature egg load and injection, or the effect of 13/24 injection alone (Table 2). However, females had higher probabilities of winning contest when they had more ready-to-lay-eggs before contests (Table 2, Fig. 4).

Discussion

The purpose of our study was to test the effect of JH manipulation on female reproductive status, aggressiveness and conflict resolution in the solitary ectoparasitoid E.

14/24 vuilleti. By measuring how JHIII injections affect oocyte maturation, we showed that JHIII did not influence mature egg load after 4h, but had a dose-dependent gonadotropic effect in females 24h post-injection. Moreover, JHIII injections and egg load interacted to affect female aggressiveness during competition for host access.

Effect of JH on females' reproductive status

In our first experiment, the reproductive status of the wasps varied after 24h according to the amount of JHIII injected in females: wasps injected with JHIII had more ready-to-lay eggs than those injected only with acetone. This result therefore suggests that JH has a gonadotropic effect in E.

vuilleti females, and was expected given that JH plays an important and diverse role in adult reproduction of many insect species by regulating vitellogenesis in the fat body and vitellogenin uptake by the ovaries (reviewed by [START_REF] Tobe | Structure and Regulation of the Corpus Allatum[END_REF][START_REF] Nijhout | Insect Hormones[END_REF][START_REF] Robinson | Juvenile hormone in adult eusocial hymenoptera: Gonadotropin and behavioral pacemaker[END_REF][START_REF] Hartfelder | Insect juvenile hormone: from "status quo" to high society[END_REF]. In Hymenoptera, JH is involved in the regulation of ovarian development in the facultatively eusocial sweat bee Megalopta genalis [START_REF] Smith | Juvenile hormone levels reflect social opportunities in the facultatively eusocial sweat bee Megalopta genalis (Hymenoptera : Halictidae)[END_REF], primitively social polistine wasps [START_REF] Barth | Juvenile hormone promotes dominance behavior and ovarian development in social wasps (Polistes annularis)[END_REF], halictine bees [START_REF] Bell | Factors controlling initiation of vitellogenesis in a primitively social bee, Lasioglossum zephyrum (Hymenoptera: Halictidae)[END_REF] and bumblebees [START_REF] Bloch | Juvenile hormone titers, juvenile hormone biosynthesis, ovarian development and social environment in Bombus terrestris[END_REF], but not in the highly social honeybee Apis mellifera (reviewed in [START_REF] Robinson | Regulation of Division of Labor in Insect Societies[END_REF][START_REF] Robinson | Juvenile hormone in adult eusocial hymenoptera: Gonadotropin and behavioral pacemaker[END_REF].

Because egg maturation was affected by JH in a dose-dependent way, we can reject the hypothesis that the physiological response to injection was an artefact related to activation of another signaling pathway such as the stress response. Finally, we found no difference in mature egg load between control and JHIII treated females 4h after injections. This is consistent with what we found in the second experiment, where contestants had the same number of mature eggs before contests, whether they were injected with JHIII or with vehicle alone. Our results therefore suggest that the egg maturation response to JH injection may happen only after 4h post-injection. Decoupling the effect of ovarian maturation and JH treatment on aggressive interactions is sometimes difficult (see [START_REF] Barth | Juvenile hormone promotes dominance behavior and ovarian development in social wasps (Polistes annularis)[END_REF] for an example). Observing E. vuilleti females fight over hosts between 1h and 4h post-injection permitted us to disentangle the effects of JHIII and mature egg load on aggressiveness and contest outcomes.

Effect of JH and Subjective Resource Value on female competition

In our second experiment, we investigated the role of JH injection and mature egg load on female aggressiveness and contest outcome. Our results support the idea that reproductive state influences behavioral decisions during fights [START_REF] Neat | Fighting and assessment in male cichlid fish: the effects of asymmetries in gonadal state and body size[END_REF][START_REF] Stokkebo | The importance of being gravid: egg load and contest outcome in a parasitoid wasp[END_REF][START_REF] Arnott | Information gathering and decision making about resource value in animal contests[END_REF]. As previously demonstrated in E. vuilleti [START_REF] Mohamad | Can subjective resource value affect aggressiveness and contest outcome in parasitoid wasps?[END_REF][START_REF] Mathiron | Let the most motivated win : resource value components affect contest outcome in a parasitoid wasp[END_REF], we found that more gravid females (i.e., females with more mature eggs) displayed more attacks and had a higher probability of winning contests. This is also consistent with game theoretical predictions that contest outcome is influenced by differences in the value that opponents place in the resource ('Subjective Resource Value', SRV; Maynard- [START_REF] Maynard-Smith | The logic of asymmetric animal contests[END_REF][START_REF] Hammerstein | The role of asymmetries in animal contests[END_REF][START_REF] Enquist | Evolution of fighting behaviour: The effect of variation in resource value[END_REF]. For instance, parasitoid females with more developed reproductive tissues are predicted to place a greater value on hosts: they are more prepared to oviposit and should therefore be more motivated to fight and have a higher probability of winning contests [START_REF] Stokkebo | The importance of being gravid: egg load and contest outcome in a parasitoid wasp[END_REF][START_REF] Brown | Aggressive contests in house crickets: size, motivation and the information content of aggressive songs[END_REF][START_REF] Brown | The payoff of fighting in house crickets: Motivational asymmetry increases male aggression and mating success[END_REF][START_REF] Dissanayake | Physiological condition and intraspecific agonistic behaviour in Carcinus maenas (Crustacea: Decapoda)[END_REF]. We therefore added evidence that the initial egg load as a component of SRV plays a main role in conflict resolution in E. vuilleti.

More interestingly, we found an interaction between JHIII injection and mature egg load on the number of aggressive behaviors displayed by females. We observed that the effect of initial egg load on aggressiveness was more pronounced when contestants were injected with JHIII. We therefore suggest that JHIII promoted female aggressiveness during fights for host access. This agrees with previous studies exploring the role of JH on agonistic behaviors in insects [START_REF] Tibbetts | The challenge hypothesis across taxa: Social modulation of hormone titres in vertebrates and insects[END_REF]. Topical application of methoprene (a JH analog) induced strong reactions to confrontation (i.e., aggression/flight behavior) in individuals of Schistocerca gregaria and Locusta migratoria [START_REF] Wiesel | Effects of Juvenile Hormone and Juvenile Hormone Analogues on the Phase Behaviour of Schistocerca gregaria and Locusta migratoria[END_REF]. [START_REF] Scott | The role of juvenile hormone in competition and cooperation by burying beetles[END_REF] made the same observation in Nicrophorus orbicollis 16/24

and Nicrophorus tomentosus: methoprene application significantly increased the number of injuries from aggressive interactions in males and females in the absence of a resource. Moreover, in the cockroach Nauphoeta cinerea, JHIII injections sustain males' aggressive posture during intra-sexual fights [START_REF] Kou | Juvenile hormone levels are increased in winners of cockroach fights[END_REF]. Here, we highlight that JH treatment upregulated aggressive behaviors associated with reproduction in E. vuilleti females, just as testosterone does in vertebrates [START_REF] Wingfield | Androgens and Mating Systems: Testosterone-Induced Polygyny in Normally Monogamous Birds[END_REF]. This is an important result because vertebrate and insect hormones have different evolutionary histories and possess different physiological backgrounds, suggesting an evolutionary convergence of endocrine effects across vertebrates and insects [START_REF] Tibbetts | The challenge hypothesis across taxa: Social modulation of hormone titres in vertebrates and insects[END_REF].

Evolution of JH functions across hymenopterans with different degrees of sociality

JH has been observed to play two important roles in life history regulation in hymenopteran societies: gonadotropin and behavioral pacemaker [START_REF] Robinson | Juvenile hormone in adult eusocial hymenoptera: Gonadotropin and behavioral pacemaker[END_REF]. Because regulation of reproduction is generally the main role of JH in adult insects [START_REF] Nijhout | Insect Hormones[END_REF], it is assumed that this was an ancestral function of this hormone in social hymenoptera. So, how the relationship between JH regulation of reproduction and JH regulation of aggressiveness has evolved across hymenopteran taxa with different degree of sociality remains an open question. Currently, two hypotheses have been advanced: i) the 'novel-or single-function hypothesis' [START_REF] Robinson | Regulation of Division of Labor in Insect Societies[END_REF][START_REF] Robinson | Juvenile hormone in adult eusocial hymenoptera: Gonadotropin and behavioral pacemaker[END_REF]Agraharia and Gadagkar, 2003) proposes that the role of JH has changed from an exclusively reproductive function in primitively eusocial species (those lacking morphologically distinct queen and worker castes), to an exclusively behavioral function in highly eusocial societies (those containing morphologically distinct castes), whereas ii) the 'split-function hypothesis' [START_REF] West-Eberhard | Wasp societies as microcosms for the study of development and evolution[END_REF] proposes that JH affects regulation of both reproduction and behavior in ancestral non-social species, but that these different effects of JH were then divided between queens and workers in more social species (i.e. regulation of reproductive maturation in egg-laying queens and behavioral pacemaker in workers). Previous studies have lent some support to both hypotheses (Agraharia and Gadagkar, 2003;[START_REF] Giray | Juvenile hormone, reproduction, and worker behavior in the neotropical social wasp Polistes canadensis[END_REF]. To the extent that JH modulates ovarian development and aggressiveness in E. vuilleti, a strict non-social hymenopteran, our work supports the split-function hypothesis, but further investigations in non-social hymenopteran species will be essential for comparative studies on the evolution of JH function.

Conclusion

By testing the role of JH on both reproductive status and aggressiveness in female E. vuilleti,

we have shown for the first time in a non-social hymenopteran parasitoid that JH has a gonadotropic effect and promotes aggressive behaviors during contests over hosts. Because JH upregulates aggressive behaviors associated with reproduction, our data support the possibility that JH in insects and testosterone in vertebrates have parallel functions, suggesting evolutionary convergence of endocrine systems.

Founding

This work was supported by the French Ministry of Research (2016-Bourse Ministérielle-44).

Figure 1 :

 1 Figure 1: Experimental apparatus (adapted from Peterson & Hardy, 1996). Three chambers (diameter =1.8 cm, depth = 0.6 cm) linked by a narrow channel (width = 0.1 cm) were made in a plastic block, decked with Plexiglas. At the start of experiments, two E. vuilleti females (one control and one test female) were simultaneously introduced into the central chamber already containing a 4 th instar larva of C. maculatus within a gelatin capsule mimicking a seed. Wasp behavior was observed from above the contest arena.

Figure 2 :

 2 Figure 2: Number of mature eggs of females injected with different doses of JHIII. a) 4h after injections, b) 24h after injections. ns: non-significant; *: P < 0.05. Data points have been horizontally displaced from their binary positions to show the numbers of observations.

Figure 3

 3 Figure 3. a) Number of attacks displayed by females treated with acetone alone (light data points, light gray line; N=25)or with JHIII (dark data points, dark curve; N=25) when fighting for a 4 th instar larva of C. maculatus. **: P < 0.01. Data points have been horizontally displaced from their binary positions to show the numbers of observations. Statistical analyses showed that effect of 'Egg load x Injection' interaction was still significant when removing data from the contest with a JHIII female displaying 22 attacks and her control opponent displaying 2 attacks (GLMM: χ²=4.87, df=1, P=0.03).

Figure 4 .

 4 Figure 4. Mean (± S.E.M) number of mature eggs in females before the contest (i.e. initial egg load) and their winning

Table 1 .

 1 Effect of initial egg load difference and injection on female agonistic behaviors.

	Factors affecting female aggressiveness	df	χ²	P
	Egg load	1	4.81	< 0.05
	Injection	1	20.34	< 0.001
	Egg load x Injection	1	7.09	0.008

P-values of significant explanatory variables are shown in bold font

Table 2 .

 2 Effect of initial egg load and injection on the winning probability of females.

	Factors affecting contest outcomes	df	χ²	P
	Egg load	1	4.62	0.03
	Injection	1	0.54	0.46
	Egg load x Injection	1	0.22	0.64

P-values of significant explanatory variables are shown in bold font

Acknowledgement

We are grateful to Charlotte Lécureuil and Joël Meunier for their advices and Fabrice Vannier for his technical assistance. We thank Dr. Mark Sheridan and 2 anonymous reviewers for helpful comments on the manuscript.


Guillaume Cayla
email: cayla.guillaume@gmail.com

René Koning

Jean Fajadet

Joel Sainsous

Didier Carrié

Simon Elhadad

Francois Tarragano

Thierry Lefevre

Sylvain Ranc

Said Ghostine

Simon El Hadad

Thierry Lefévre

Said Ghostine j Philippe Garot

Frédéric Marco

Luc Maillard

Pascal Motreff

Hervé Le Breton

Percutaneous coronary interventions with the Absorb Bioresorbable vascular scaffold in real life 1-year results from the FRANCE ABSORB registry

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

décès cardiovasculaire, infarctus du myocarde et de revascularisation de la lésion cible à 1 an. Les critères secondaires étaient la thrombose de stent et la revascularisation du vaisseau cible à un an.

Résultats. -Un total de 2072 patients ont été inclus dans 86 centres, l'âge moyen était de 55 ± 11 ans; 80 % étaient des hommes. L'indication était un syndrome coronarien aigu (SCA) dans 49 % des cas. Des pré et post dilatations ont été réalisées respectivement dans 93 % et 83 % des lésions. Après un suivi de 1 an, le critère principal de jugement était de 3,9 % ; le taux de thrombose de BVS était de 1,5 % et le taux de revascularisation du vaisseau cible était de 3,3 %. En analyse multivariée, le diabète et la longueur totale des BVS > 30 mm étaient indépendamment associées à la survenue du critère principal de jugement alors que l'anticoagulation orale et la longueur totale des BVS > 30 mm étaient indépendamment associées à la survenue d'une thrombose de stent.

Conclusions. -Les BVS ont été implantés dans une population relativement jeune, dont la moitié avait un SCA. Les taux de pré et post dilatation étaient élevés. Les résultats cliniques à un an étaient acceptables.

KEYWORDS

Bioresorbable scaffolds;

Percutaneous coronary intervention;

Acute coronary syndrome

MOTS CLÉS

Stent bioresorbable ;

Angioplastie coronaire ; Syndrome coronaire aigu Abbreviations: ACS, acute coronary syndrome; BVS: bioresorbable vascular scaffold; CE, Conformité Européene (European Conformity); DAPT: dual antiplatelet therapy; MACE: major adverse cardiac events; PCI: percutaneous coronary intervention.

Background

Bioresorbable vascular scaffold (BVS) technology (Absorb BVS; Abbott Vascular, Santa Clara, CA, USA) was developed to overcome the limitations of drug-eluting stents, such as the development of neoatherosclerosis, vessel vasomotricity limitation and late thrombosis events [START_REF] Serruys | From metallic cages to transient bioresorbable scaffolds: change in paradigm of coronary revascularization in the upcoming decade?[END_REF].

The current generation of Absorb BVS consists of a balloon-expandable scaffold with a polymer backbone of poly-L-lactic acid coated with an amorphous matrix of poly-D-and poly-L-lactic acid polymers, covered by the antiproliferative drug everolimus [START_REF] De Pommereau | Bioresorbable vascular scaffolds: Time to absorb past lessons or fade away?[END_REF]. The process of degradation of the scaffold is by hydrolysis, and full resorption of the device is expected approximately 3 years after implantation [START_REF] Brie | Bioresorbable scaffold -A magic bullet for the treatment of coronary artery disease?[END_REF].

The Absorb BVS was approved by the Food and Drug Administration in 2016, and obtained a Conformité Européene (CE; European Conformity) mark in 2011.

Randomized trials and meta-analyses have indicated a potentially increased risk of scaffold thrombosis using this new technology compared with an everolimus-eluting stent [START_REF] Ellis | Everolimus-Eluting Bioresorbable Scaffolds for Coronary Artery Disease[END_REF][START_REF] Kang | Stent Thrombosis With Drug-Eluting Stents and Bioresorbable Scaffolds: Evidence From a Network Meta-Analysis of 147 Trials[END_REF][START_REF] Serruys | A bioresorbable everolimus-eluting scaffold versus a metallic everolimus-eluting stent for ischaemic heart disease caused by de-novo native coronary artery lesions (ABSORB II): an interim 1-year analysis of clinical and procedural secondary outcomes from a randomised controlled trial[END_REF][START_REF] Wykrzykowska | Bioresorbable Scaffolds versus Metallic Stents in Routine PCI[END_REF]. The reason for the increase is not fully understood, but recent analyses have suggested that the quality of the implantation procedure (predilatation, correct sizing and postdilatation) may improve the efficacy and safety of the scaffold [START_REF] Stone | Effect of Technique on Outcomes Following Bioresorbable Vascular Scaffold Implantation: Analysis From the ABSORB Trials[END_REF].

When Absorb BVS technology was introduced in France in 2013, the Working Group for Interventional Cardiology of the French Society of Cardiology decided to conduct a prospective registry, aiming to include all patients with at least one Absorb BVS implanted, and with predilatation and postdilatation performed as frequently as possible. Such an exhaustive analysis of all Absorb BVS implantation procedures in French centres was also requested by the French Agence Nationale de Sécurité des Médicaments (ANSM; French National Agency for the Safety of Medicines and Health Products), to reflect the experiences and clinical outcomes of patients in a real-life setting. Accordingly, the nationwide FRANCE ABSORB registry was initiated in 2014; here we present the 1-year outcomes.

Methods

Study design and patient population

The FRANCE ABSORB registry (ClinicalTrial.gov unique identifier NCT02238054) is a prospective observational multicentre study designed to evaluate the clinical efficacy and safety of the Absorb BVS used in a real-life setting in patients presenting with de novo lesion(s).

Clinical and angiographic patient data were recorded anonymously in an electronic Clinical Research File, which was exported to an independent database under the responsibility of the French Cardiac Society.

All patients provided written informed consent, including consent for anonymous processing of their data. The registry was approved by the French National Agency for the Safety of Medicines and Health Products. The study was conducted in accordance with the Declaration of Helsinki.

Study procedure

Percutaneous coronary intervention (PCI) was performed according to current clinical practice standards.

Implantations were performed without specific inclusion/exclusion criteria, but respecting the CE certification and the manufacturer's indications. Centres were recommended not to implant the Absorb BVS in highly calcified lesions, left main coronary artery lesions, bifurcation lesions with a side branch > 2 mm in diameter, in-stent restenosis lesions, bypass graft lesions or vessels with excessive tortuosity.

Procedural recommendations were as follows: predilatation, optimal Absorb BVS sizing, step-by-step optimal Absorb BVS deployment, systematic postdilatation with a non-compliant balloon (with a diameter up to 0.25 mm larger than the Absorb BVS diameter). When possible, the use of optical coherence tomography in the early implantation experience was encouraged.

The timing and type of antiplatelet therapy (aspirin, clopidogrel, prasugrel, ticagrelor) were according to each hospital's standard procedures. The use of glycoprotein IIb/IIIa inhibitors was left to the physicians' discretion. Dual antiplatelet therapy (DAPT) was recommended for a period of 12 months.

Data management

An independent clinical events committee, composed of physicians provided with all necessary and available data, adjudicated all major cardiovascular events and protocol endpoints. Data from 207 patients (10% of the population), selected to be representative of the size and geographical location of the centres, were monitored by clinical research assistants from the French Society of Cardiology.

Endpoints

The primary endpoint was the occurrence of major adverse cardiac events (MACE) at 1 year, including death from cardiac causes, myocardial infarction (defined according to the third universal definition [START_REF] Thygesen | Third universal definition of myocardial infarction[END_REF]) and target lesion revascularization. Secondary outcomes were rate of scaffold thrombosis (definite/probable/possible), defined according to the Academic Research Consortium, at 1 year [START_REF] Cutlip | Clinical end points in coronary stent trials: a case for standardized definitions[END_REF], and rate of target vessel revascularization at 1 year. The FRANCE ABSORB registry is on-going, and outcomes will be evaluated yearly, over a 5-year follow-up period.

Study supervision

The study was initiated by the Interventional Working Group of Cardiology (GACI) of the French Society of Cardiology. The scientific study committee was responsible for the development of the protocol and the writing of the manuscript. The committee had unrestricted access to all study data.

Statistical analysis

The data were analysed descriptively. Categorical data are presented as numbers and percentages, and continuous data as means and standard deviations (or medians and interquartile ranges when skewed).

For categorical data, differences between groups were tested using the χ² test or Fisher's exact test, as appropriate. For continuous data, differences between groups were tested using Student's t test (or the Mann-Whitney non-parametric test in case of skewed distribution).

Multivariable analyses were performed to identify factors associated with MACE or stent thrombosis occurrence during the first 12 months, using Cox proportional hazards models. Population and procedure characteristics that were associated with MACE or stent thrombosis with P < 0.20 in unadjusted analyses were included in the multivariable analysis.

All data handling and analyses were performed using Stata statistical software, release 10 (StataCorp, College Station, TX, USA).

Results

Patients

From September 2014 to April 2016, 2072 patients receiving at least one Absorb BVS were prospectively and consecutively included at 86 sites in France (including la Reunion and New Caledonia); Table 1 summarizes their baseline demographics. The mean age was 55 ± 11 years, and 80% of patients were men. Acute coronary syndromes (ACSs) accounted for 49% of the indications, and 17% of subjects had ST-segment elevation myocardial infarction. A quarter of the population had undergone a previous PCI, and 16% had a history of myocardial infarction. Current smoking was found in 41% of patients, hypertension in 43% of patients, but diabetes in only 16% of patients.

The angiographic characteristics of the lesions treated with the Absorb BVS are shown in Table 2.

Among lesions for which the type was known, 44% were type B2 or C, according to the American Heart Association/American College of Cardiology classification. Most (61%) were located on the left anterior descending artery or the diagonal branch. The diameter of the Absorb BVS was 2.5 mm for 24.2% of implants. Contrary to operator recommendations, Absorb BVSs were used to treat 13 lesions on the left main coronary artery, 22 in-stent restenosis lesions and two lesions in a bypass graft (Appendix Table A.1).

Procedural data

Details were recorded for 2818 Absorb BVSs implanted in 2072 patients and 2502 lesions. During the index procedure, the radial approach was used in 91% of patients. Unfractionated heparin, low-molecularweight heparin and bivalirudin were used in 80%, 18% and 1% of cases, respectively. The mean number of Absorb BVSs per patient was 1.35 ± 0.68; 26.6% of patients received more than one Absorb BVS. In 18.5% of patients, a metallic drug-eluting stent or bare-metal stent was placed in another significant lesion.

As recommended by the study protocol, predilatation was performed in 93% of lesions. Among 2818 Absorb BVSs implanted, the mean length was 20.7 ± 5.7 mm, with a mean diameter of 3.05 ± 0.39 mm. 2. To optimize implantation, optical coherence tomography was used for 15%, and postdilatation was performed in 83% of the lesions treated with the Absorb BVS.

Distributions of length and diameter of scaffolds are shown in Table

Angiographic procedural success for all lesions treated with the Absorb BVS was achieved in 99.2% of patients. The median postprocedural stay was 2 days (interquartile range 2-3 days). Nearly all patients (98%) were discharged under DAPT (clopidogrel 42.9%; ticagrelor 36%; prasugrel 21%). Notably, 2.4% of patients were treated with an oral anticoagulant (vitamin K antagonist or non-vitamin K antagonist oral anticoagulant).

Clinical endpoints

One-year events are summarized in Table 3. Complete follow-up data at 1 year were available for 2039 patients (98.4%). After 1 year, a primary endpoint event (cardiovascular death, myocardial infarction or target lesion revascularization) had been observed in 41/2072 patients (3.9%). Cardiovascular death at 1 year occurred in 14/2072 patients (0.7%). Myocardial infarction and target lesion revascularization occurred in 2.6% (n = 54) and 2.4% (n = 49) of patients, respectively. Among secondary endpoints, the rate of definite/probable stent thrombosis was 1.5% (31/2072) (Fig. 1 and Fig. 2). The target vessel revascularization rate was 3.3% (68/2072).

Factors associated with occurrence of MACE and scaffold thrombosis

Multivariable analyses of factors associated with occurrence of MACE or definite/probable scaffold thrombosis at 1 year are presented in Tables 4 and5 and Appendix Table A.2. Factors significantly associated with scaffold thrombosis were oral anticoagulation and the total Absorb BVS length per patient.

Discussion

In the FRANCE ABSORB registry, the use of the Absorb BVS in a selected population, with a large proportion of predilatation and postdilatation, was associated with a low rate of MACE and acceptable BVS thrombosis rates at 1 year.

FRANCE ABSORB registry population

Patients included in the FRANCE ABSORB registry are selected patients, as indicated by the low mean age compared with other studies [START_REF] Nef | Everolimus-eluting bioresorbable scaffolds in patients with coronary artery disease: results from the German-Austrian ABSORB RegIstRy (GABI-R)[END_REF][START_REF] Stone | 1-year outcomes with the Absorb bioresorbable scaffold in patients with coronary artery disease: a patient-level, pooled meta-analysis[END_REF]. There may be particular advantages to using BVSs in younger patients (e.g. restoration of pulsatility, vasomotricity or the possibility of surgical lesion bypass) [START_REF] Serruys | From metallic cages to transient bioresorbable scaffolds: change in paradigm of coronary revascularization in the upcoming decade?[END_REF]. Half of the registry population presented with ACS, and most of the patients had single-vessel disease and normal left ventricular function, which probably also reflects the selection of patients and lesions.

Implantation and preparation of the lesion

The implantation success rate was high, despite the 150 µm strut thickness of the current Absorb BVS generation. The high success rate might be explained by closer adherence to current recommendations for the use of the Absorb BVS (no severe calcifications, no excessive tortuosity, no left main coronary artery, no bifurcation, no restenosis, no saphenous vein graft and strict lesion selection). Optimal Absorb BVS expansion requires adequate lesion preparation, especially in more complex lesions. Current recommendations highlight the importance of predilatation and high-pressure postdilatation to achieve optimal scaffold expansion and, possibly, better clinical outcomes [START_REF] Stone | Effect of Technique on Outcomes Following Bioresorbable Vascular Scaffold Implantation: Analysis From the ABSORB Trials[END_REF][START_REF] Puricel | Bioresorbable Coronary Scaffold Thrombosis: Multicenter Comprehensive Analysis of Clinical Presentation, Mechanisms, and Predictors[END_REF]. Here, we report high predilatation and postdilatation rates (93.0% and 82.7%, respectively).

Efficacy outcomes and scaffold thrombosis

Low rates of MACE and target lesion failure were found in the FRANCE ABSORB registry, which is in agreement with data from several randomized trials and meta-analyses [START_REF] Ellis | Everolimus-Eluting Bioresorbable Scaffolds for Coronary Artery Disease[END_REF][START_REF] Serruys | A bioresorbable everolimus-eluting scaffold versus a metallic everolimus-eluting stent for ischaemic heart disease caused by de-novo native coronary artery lesions (ABSORB II): an interim 1-year analysis of clinical and procedural secondary outcomes from a randomised controlled trial[END_REF][START_REF] Wykrzykowska | Bioresorbable Scaffolds versus Metallic Stents in Routine PCI[END_REF][START_REF] Stone | 1-year outcomes with the Absorb bioresorbable scaffold in patients with coronary artery disease: a patient-level, pooled meta-analysis[END_REF][START_REF] Chevalier | Four-year follow-up of the randomised comparison between an everolimus-eluting bioresorbable scaffold and an everolimus-eluting metallic stent for the treatment of coronary artery stenosis (ABSORB II Trial)[END_REF][START_REF] Kereiakes | Efficacy and Safety of the Absorb Everolimus-Eluting Bioresorbable Scaffold for Treatment of Patients With Diabetes Mellitus: Results of the Absorb Diabetic Substudy[END_REF][START_REF] Serruys | Comparison of an everolimus-eluting bioresorbable scaffold with an everolimus-eluting metallic stent for the treatment of coronary artery stenosis (ABSORB II): a 3 year, randomised, controlled, single-blind, multicentre clinical trial[END_REF][START_REF] Sorrentino | Everolimus-Eluting Bioresorbable Scaffolds Versus Everolimus-Eluting Metallic Stents[END_REF].

The 1-year rate of scaffold thrombosis reported in the FRANCE ABSORB study is in line with reports from other European registries (GABI-R [START_REF] Nef | Everolimus-eluting bioresorbable scaffolds in patients with coronary artery disease: results from the German-Austrian ABSORB RegIstRy (GABI-R)[END_REF], GHOST EU [START_REF] Capodanno | Percutaneous coronary intervention with everolimus-eluting bioresorbable vascular scaffolds in routine clinical practice: early and midterm outcomes from the European multicentre GHOST-EU registry[END_REF] and ABSORB UK [START_REF] Baumbach | Acute and one-year clinical outcomes following implantation of bioresorbable vascular scaffolds: the ABSORB UK Registry[END_REF]) and randomized studies [START_REF] Ellis | Everolimus-Eluting Bioresorbable Scaffolds for Coronary Artery Disease[END_REF][START_REF] Serruys | A bioresorbable everolimus-eluting scaffold versus a metallic everolimus-eluting stent for ischaemic heart disease caused by de-novo native coronary artery lesions (ABSORB II): an interim 1-year analysis of clinical and procedural secondary outcomes from a randomised controlled trial[END_REF]. There has been concern about excess risk of BVS thrombosis after implantation. As with metallic stents [START_REF] Cayla | Clinical, angiographic, and genetic factors associated with early coronary stent thrombosis[END_REF], the mechanisms underlying scaffold thrombosis are multifactorial [START_REF] Yamaji | Mechanisms of Very Late Bioresorbable Scaffold Thrombosis: The INVEST Registry[END_REF], and may be related to the technique of implantation, persistence of uncovered struts, strut malapposition or underexpansion of the scaffold [START_REF] Kang | Stent Thrombosis With Drug-Eluting Stents and Bioresorbable Scaffolds: Evidence From a Network Meta-Analysis of 147 Trials[END_REF]. Rates of probable and definite scaffold thrombosis of 1.8% at 30 days and 3% at 12 months were reported recently in a large multicentre registry of 1305 all-comer patients who received a total of 1870 Absorb BVSs [START_REF] Puricel | Bioresorbable Coronary Scaffold Thrombosis: Multicenter Comprehensive Analysis of Clinical Presentation, Mechanisms, and Predictors[END_REF]. In this study, suboptimal postprocedural angiographic results were associated with an increase in risk of BVS thrombosis. The in-BVS thrombosis rate decreased over time with improvement of implantation techniques. A similar improvement over time was observed in the GABI-R registry [START_REF] Nef | Everolimus-eluting bioresorbable scaffolds in patients with coronary artery disease: results from the German-Austrian ABSORB RegIstRy (GABI-R)[END_REF]. Higher definite scaffold thrombosis rates with the Absorb BVS compared with the XIENCE stent (Abbott Vascular, Santa Clara, CA, USA) were recently reported in the ABSORB 2 and ABSORB 3 trials after 4 and 3 years of follow-up, respectively [START_REF] Chevalier | Four-year follow-up of the randomised comparison between an everolimus-eluting bioresorbable scaffold and an everolimus-eluting metallic stent for the treatment of coronary artery stenosis (ABSORB II Trial)[END_REF][START_REF] Kereiakes | 3-Year Clinical Outcomes With Everolimus-Eluting Bioresorbable Coronary Scaffolds: The ABSORB III Trial[END_REF].

Factors associated with the occurrence of MACE and scaffold thrombosis

We found that diabetes mellitus, total Absorb BVS length > 30 mm and oral anticoagulation were associated with the occurrence of MACE or scaffold thrombosis. A long Absorb BVS length was associated with more adverse events than shorter lengths. The management of long or multiple lesions using the Absorb BVS is particularly challenging. The negative association between scaffold length and scaffold thrombosis was recently described in a subgroup analysis from the GHOST registry [START_REF] Geraci | Bioresorbable Everolimus-Eluting Vascular Scaffold for Long Coronary Lesions: A Subanalysis of the International, Multicenter GHOST-EU Registry[END_REF]. An increased risk of restenosis and cardiovascular events in patients with diabetes has been described for metallic drug-eluting stents [START_REF] Stone | Differential clinical responses to everolimus-eluting and Paclitaxel-eluting coronary stents in patients with and without diabetes mellitus[END_REF], and has also been observed more recently using the Absorb BVS [START_REF] Capodanno | Percutaneous coronary intervention with everolimus-eluting bioresorbable vascular scaffolds in routine clinical practice: early and midterm outcomes from the European multicentre GHOST-EU registry[END_REF].

An association between scaffold thrombosis and oral anticoagulation has not been reported before.

The choice and duration of antiplatelet therapy after implantation of the Absorb BVS is difficult [START_REF] Valgimigli | ESC focused update on dual antiplatelet therapy in coronary artery disease developed in collaboration with EACTS: The Task Force for dual antiplatelet therapy in coronary artery disease of the European Society of Cardiology (ESC) and of the European Association for Cardio-Thoracic Surgery (EACTS)[END_REF],

particularly for patients who need oral anticoagulation in association with antiplatelet agents. Our data indicate that implantation of the Absorb BVS in patients with a high bleeding risk, including patients under an oral anticoagulant, should probably be discouraged. This is in line with the recent consensus document from the European Society of Cardiology/European Association for Cardio-Thoracic Surgery, which does not recommend the use of the Absorb BVS in patients who cannot tolerate an extended duration of DAPT or who require oral anticoagulation [START_REF] Byrne | Report of an ESC-EAPCI Task Force on the evaluation and use of bioresorbable scaffolds for percutaneous coronary intervention: executive summary[END_REF].

Study limitations

As FRANCE ABSORB was a prospective registry, the enrolment of the patients was not randomized, and therefore some selection bias cannot be excluded. All Absorb BVS implantations in France had to be included in the registry during the inclusion period. However, it cannot be excluded that some subjects with the Absorb BVS implanted in France during this period were not included in the registry. As there was no control group, a comparison with other treatment options is not possible. We acknowledge that the details of the DAPT regimen at 1 year were not available, and that a core laboratory was not used for the angiographic analysis.

Conclusions

The FRANCE ABSORB registry is one of the largest registries conducted with the Absorb BVS. High proportions of young patients and patients with ACS were included. Current recommendations were followed to a large degree, with high predilatation and postdilatation rates. Absorb BVS implantation was associated with low rates of 1-year MACE. BVS thrombosis rates were acceptable at 1 year, although the results may be different in a non-selected population that includes older patients. The scheduled 5-year follow-up in the whole population is justified by the indication of possible late scaffold thrombosis. a MI: according to the third universal definition of MI, all types (1, 2, 3, 4 and 5) were considered.

b Target vessel MI: all infarcts that could not clearly be attributed to a vessel other than the target vessel were considered related to the target vessel.

c Target lesion revascularization was defined as any repeat revascularization of the target lesion for restenosis or other complications of the target lesion; all target lesion revascularizations clinically indicated and not clinically indicated were considered.

d Target vessel revascularization was defined as any repeat revascularization of any segment of the target vessel.

e Cardiac death or MI or target lesion revascularization.

f Cardiac death or target vessel MI or target lesion revascularization.

g Cardiac death or target vessel MI or target vessel revascularization or target lesion revascularization.

h Any death or any MI or any revascularization.

Figure 1 .

 1 Figure 1. Occurrence of bioresorbable vascular scaffold (BVS) definite/probable scaffold thrombosis during 1-year (1Y) follow-up.

Figure 2 .

 2 Figure 2. Occurrence of bioresorbable vascular scaffold (BVS) definite/probable scaffold thrombosis during 1-year (1Y) follow-up, according to total length of scaffold.

  Figure 1

Figure 2

 2 Figure 2

Table 1

 1 Baseline characteristics of the study cohort (n = 2072).

	Age (years)

Data are expressed as mean ± standard deviation or number (%). ACS: acute coronary syndrome; BVS: bioresorbable vascular scaffold; CABG: coronary artery bypass graft; DAPT: dual antiplatelet therapy; LMWH: low-molecular-weight heparin; LVEF: left ventricular ejection fraction; MI: myocardial infarction; NOAC: non-vitamin K antagonist oral anticoagulant; NSTEMI: non-ST-segment elevation myocardial infarction; PCI: percutaneous coronary intervention; STEMI: ST-segment elevation myocardial infarction; UFH: unfractionated heparin; VKA: vitamin K antagonist. a For index procedure.

Table 2

 2 Characteristics of lesions treated with the Absorb bioresorbable vascular scaffold (n = 2502),

	and procedural data.
	Lesion localization
	LAD/Diag

artery or diagonal branch; PDA: posterior descending artery; PLV: posterior left ventricular artery; RCA: right coronary artery; TIMI: Thrombolysis in Myocardial Infarction.

Table 3

 3 One-year clinical events (n = 2072).

	Number of clinical events	%

Table 4

 4 Factors associated with major adverse cardiac events at 1 year: multivariable Cox regression.

	Univariate analysis		Multivariable analysis
	Hazard ratio	95% CI	P	Hazard ratio	95% CI	P

a For index procedure. b Fisher's exact test.

Table 5

 5 Factors associated with bioresorbable vascular scaffold thrombosis (definite and probable) occurrence at 1 year: multivariable Cox regression. : bioresorbable vascular scaffold; CI: confidence interval; Gp: glycoprotein; LVEF: left ventricular ejection fraction; MI: myocardial infarction; NOAC: nonvitamin K antagonist oral anticoagulant; NSTEMI: non-ST-segment elevation myocardial infarction; PCI: percutaneous coronary intervention; PSP: predilatation, sizing and postdilatation; STEMI: ST-segment elevation myocardial infarction; UFH: unfractionated heparin; VKA: vitamin K antagonist..

	Univariate analysis		Multivariable analysis
	Hazard ratio	95% CI	P	Hazard ratio 95% CI	P

BVS

a For index procedure. b Fisher's exact test.

a Achievement of final in-scaffold stenosis of < 30% and TIMI flow grade 3.

Acknowledgements

We thank Vincent Bataille for performing the statistical analysis, and Geneviève Mulak, Elodie Drouet and Nicole Naccache from the French Society of Cardiology for their assistance. We also thank Pr H.

Eltchaninoff, Pr C. Bauters and Dr P. Meyer for their contribution to the event assessment committee.

R. K. Consulting fees from the company

Abbott Vascular. P. M. Consulting fees from the companies Abbott Vascular and Terumo. H. L. B. Consultant to the company Abbott. Speaker fees from the companies Edwards Lifesciences and Medtronic.

The other authors declare that they have no conflicts of interest concerning this article.

Sources of funding

The study was funded by Abbott; the sponsor had no involvement in the study design, data collection, data analysis or writing of the report.

Disclosure of interest


Hassan Allouchi

René Ceolin

Alain Gueiffier

Ivo B Rietveld
email: ivo.rietveld@univ-rouen.fr

A Network of Weak Hydrogen Bonds in the Crystal Structure of Tetrazepam Un Réseau de Liaisons Hydrogène Faibles dans la Structure Cristalline du Tétrazepam

Keywords: Tetrazepam, crystal structure, weak hydrogen bonds Mots-clés Tétrazepam, structure cristalline, liaisons hydrogène faibles

) Å 3 at room temperature (293 K) with Z = 4 molecules in the unit-cell. A network of weak hydrogen bonds involving aliphatic hydrogen atoms plays an important role in the formation of this structure.

Résumé

) Å, et Z = 4 molécules par maille élémentaire à température ambiante (293 K). Un réseau de liaisons hydrogène faibles avec des hydrogènes aliphatiques joue un rôle important dans la formation de cette structure.

Introduction

Tetrazepam (C16H17ClN2O, IUPAC name: 7-chloro-5-(cyclohex-1-enyl)-1,3-dihydro-1methyl-2H-1,4-benzodiazepin-2-one, M= 288.77 g mol -1 ) is a benzodiazepine derivative formerly prescribed for its muscle relaxant properties and the management of anxiety disorders. However, due to side effects such as allergic hypersensitivity reactions, it has been suspended from the market in the European Union since 2013 [START_REF] Poy-Vega | Requiem for tetrazepam[END_REF].

Although it has been patented about fifty years ago [2], its structural details through Xray diffractometry surprisingly have been found wanting. Therefore, the crystal structure of tetrazepam was solved using single-crystal X-ray diffractometry and the results are presented in the following.

Materials and methods

Crystal preparation

A sample of tetrazepam powder of medicinal grade was kindly supplied by Daiichi Sankyo France SAS and single crystals were obtained by slowly evaporating solutions in methanol at room temperature.

Single-crystal measurements, structure determination and refinement

Intensities were collected through ω scans at 293 K from a crystal mounted on a Bruker-Nonius κ-CCD diffractometer, equipped with a CCD camera as an area detector using a graphite-monochromator and Mo-Kα radiation (λ = 0.71073 Å) from a fine-focus sealed tube. The unit cell determination, refinement, and data collection have been carried out using the Collect [3] and Dirax programs [START_REF]Dirax/lsq program[END_REF]. Data reduction was carried out with the EvalCCD program [START_REF] Evalccd Program | [END_REF]. A semi-empirical absorption correction was carried out using the SADABS program [START_REF] Sheldrick | SADABS[END_REF]. The positions of non-hydrogen atoms have been determined and refined with the SHELXS program [START_REF] Sheldrick | A short history of SHELX[END_REF]. They were first refined isotropically and then anisotropically by a full-matrix least squares method based on F 2 using the SHELXe program [8]. The hydrogen atoms were introduced on positions determined by the geometrical model of the hybridized carbon atoms to which they are linked with isotropic atomic displacement parameters set proportional to those of the linked atoms by a factor of 1.2. Geometry and proportional constraints were kept constant (rigid group) reducing the refinement to that of the coordinates and atomic displacement parameters of the atoms to which the hydrogens are linked.

Results and discussion

Tetrazepam was found to crystallize at 293 K in the monoclinic system, space group P21/c, with lattice parameters a = 12.7386(7) Å, b = 11.3774(7) Å, c = 10.3084(7) Å, β= 103.175(5)°, Vcell = 1454.69(16) Å, and Z = 4 molecules per unit-cell. Crystallographic information is compiled in Table 1 and the fractional atomic coordinates of the non-hydrogen atoms are listed in Table 2. Other data (bond lengths, bond angles, anisotropic displacement parameters, hydrogen-atoms coordinates, torsion angles) can be found in Tables S1 to S4 of the Supplementary Information. The crystal structure in the form of a CIF has been submitted to the Cambridge Crystallographic Data Centre (CCDC) with the deposition number CCDC 1857121. The CIF contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk/data_request/cif. The molecular structure of tetrazepam with labels for the non-hydrogen atoms is presented in Figure 1. In the monoclinic packing of the tetrazepam crystals, the molecules are held together by a complicated network of weak C-H•••O and C-H•••Cl hydrogen bonds [START_REF] Desiraju | The C-H•••O Hydrogen Bond in Crystals: What Is It ?[END_REF][START_REF] Steiner | Distinction between the weak hydrogen bond and the van der Waals interaction[END_REF][START_REF] Steiner | C-H•••O hydrogen bonding in crystals[END_REF] whose distances and angles are listed in Table 3 and compared to values in the literature [START_REF] Jeffrey | Cooperative aspects of hydrogen bonding in carbohydrates[END_REF][START_REF] Taylor | Comparison of X-ray and Neutron Diffraction Results for the N-H•••O=C Hydrogen Bond[END_REF]. Bonding analysis shows that intra-and intermolecular hydrogen bonds occur. One of the more important weak C-H•••O hydrogen bonds form centrosymmetric dimers shown in Figure 2. These dimers stack and form layers parallel to the bc plane, as shown in Figure 3. Although the European Community decided in 2006 through the REACH regulation [START_REF]the European Parliament and of the Council of 18 December 2006, concerning the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH)[END_REF] to "… generate information on substances and their uses." and to demand that "All available and relevant information on substances on their own, ... should be collected…", basic physicochemical properties such as crystal structures remain unknown for several older active pharmaceutical ingredients (API). In this context, the crystal structure of tetrazepam has been determined at room temperature and found to be monoclinic. Even if this API has been withdrawn from the European market, the present crystal structure can now be used for unambiguous characterization of the molecule in the solid state through X-ray powder diffraction.

Tables Table 1. Crystal data and structure refinement for tetrazepam at 293 K

Figure 1 .

 1 Figure 1. Tetrazepam molecule with labeled non-hydrogen atoms in the asymmetric unit. Pink = hydrogen atoms. Molécule de tétrazepam avec la numérotation des atomes (à l'exception des atomes d'hydrogène) dans l'unité asymétrique. Rose = atomes d'hydrogène.

Figure 2 .

 2 Figure 2. Hydrogen-bonded tetrazepam dimers. The dimers are formed by two weak C-H•••O hydrogen bonds, C(10)-H(10A)•••O(1), symbolized by the violet lines. The unit-cell is drawn in blue. Dimère de tétrazepam formé par liaisons hydrogène. Les liaisons hydrogène C-H•••O faibles sont symbolisées par les segments violets. La maille élémentaire est représentée en bleu.

Figure 3 .

 3 Figure 3. Monoclinic packing of the tetrazepam dimers parallel to the bc plane. View along cell axis b. The unit-cell is shown in blue. Empilement monoclinique des dimères de tétrazepam parallèlement au plan bc. Vue selon l'axe b de la maille élémentaire. Celle-ci est représentée en bleu.

  Largest diff. peak and hole 0.566 and -0.467 e.Å-3

Table 1 .

 1 Données cristallines et affinement de la structure du tétrazepam à 293 K

	Empirical formula	C16H17ClN2O

Table 2 . Fractional atomic coordinates (× 10 4 ) and equivalent isotropic displacement parameters (Å 2 × 10 3 ) for the non-hydrogen atoms of Tetrazepam aTable 2

 22 U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

	. Coordonnées atomiques réduites (× 10 4 ) et paramètres d'agitation thermique isotrope (Å 2 × 10 3 ) des atomes du Tetrazepam a
	x		y	z	U(eq) b
	Cl(1)	1464(1)	-929(1)	7813(1)	57(1)
	O(1)	963(2)	1738(2)	689(2)	60(1)
	N(1)	1607(2)	424(2)	2312(2)	39(1)
	N(2)	2441(2)	2617(2)	3686(2)	39(1)
	C(1)	1274(2)	1526(2)	1873(3)	40(1)
	C(2)	1335(2)	2447(2)	2931(3)	39(1)
	C(3)	2838(2)	1819(2)	4535(2)	33(1)
	C(4)	2230(2)	736(2)	4737(2)	30(1)
	C(5)	1658(2)	80(2)	3654(2)	32(1)
	C(6)	1111(2)	-926(2)	3888(3)	40(1)
	C(7)	1070(2)	-1258(2)	5161(3)	42(1)
	C(8)	1592(2)	-577(2)	6215(3)	36(1)
	C(9)	2188(2)	397(2)	6025(2)	32(1)
	C(10)	1541(3)	-482(3)	1288(3)	57(1)
	C(11)	3933(2)	2015(2)	5382(2)	38(1)
	C(12)	4536(2)	1144(2)	6010(3)	45(1)
	C(13)	5625(2)	1305(3)	6922(4)	66(1)
	C(14)	5833(4)	2599(4)	7287(5)	105(2)
	C(15)	5480(3)	3402(3)	6234(5)	94(2)
	C(16)	4317(3)	3273(3)	5503(4)	65(1)
	a e.s.d.'s in parentheses

b

Table 3 . Hydrogen bonds of the monoclinic tetrazepam packing at 293 K (distances d in Å and angles in °)

 3 Symmetry transformations used to generate equivalent atoms (opérateurs de symétrie utilisés pour engendrer les atomes équivalents): #1: x,-y+1/2, z+1/2. #2: -x, y-1/2,-z+1/2. #3: x, y, z-1. #4:-x,-y,-z.

	Liaisons hydrogène de l'empilement monoclinique du tetrazepam à 293 K (distances d en Å et

a from references

[START_REF] Jeffrey | Cooperative aspects of hydrogen bonding in carbohydrates[END_REF]

and

[START_REF] Taylor | Comparison of X-ray and Neutron Diffraction Results for the N-H•••O=C Hydrogen Bond[END_REF]

.

Conflicts of interest

No conflicts of interest to declare.