domain: shs shs

text:
Evelyne Bouteyre

LPCPP Pauline Duval

Mariel Pietri

LPCPP Evelyne Bouteyre

Children's Physical Proximity to Interparental Conflict: Resilient Process and Retrospective Perceptions of Parent-Child Relationships

Keywords: Insecurity, direct exposure, young adults, positive adjustment, child witness Children's Physical Proximity to parental Conflict 3 Children

whether they are published or not. The documents may come

that this form of resilience is the subject of numerous definitions which unfold in successive waves, each marking the evolution of the concept [START_REF] Ionescu | Résiliences. Ressemblances dans la diversité[END_REF]. In the context of children who are witnesses to, or victims of, parental conflict, the following definition appears appropriate: resilience refers to successful adaptation despite being confronted with traumatic life events or a life marked by chronic adversity [START_REF] Ionescu | Traité de résilience assistée[END_REF].

Although research has established that children do not need to be directly exposed to interparental conflict to be affected by it, the impact of their actual physical presence has seldom been considered in studies of the short-, medium-or long-term effects of this exposure [START_REF] Fantuzzo | Children's direct exposure to types of domestic violence crime: A population-based investigation[END_REF][START_REF] Latzman | Children's exposure to intimate partner violence: A systematic review of measurement strategies[END_REF][START_REF] Ovaere | L'impact de la violence conjugale sur les enfants[END_REF].

Effects of Interparental Conflict on Children

Authors investigating the short-and medium-term effects of destructive interparental conflict have clearly demonstrated social maladjustment in children and adolescents [START_REF] Fosco | Making sense of family violence: Implications of children's appraisals of interparental aggression for their short-and longterm functioning[END_REF], characterized by a high frequency of internalizing and externalizing symptoms [START_REF] Bradford | When there is conflict: Interparental conflict, parent-child conflict, and youth problem behaviors[END_REF][START_REF] Kaczynski | Marital conflict, maternal and paternal parenting, and child adjustment: A test of mediation and moderation[END_REF][START_REF] Krishnakumar | Youth perceptions of interparental conflict, ineffective parenting, and youth problem behaviors in European-American and African-American families[END_REF]. However, rather than explore it long-term effects, authors have tended to focus on how this conflict may be replicated in children's subsequent romantic relationships [START_REF] Suzuki | The experiences of adults exposed to intimate partner violence as children: An exploratory qualitative study of resilience and protective factors[END_REF]. Research has nonetheless shown that young adults who were exposed to scenes of interparental conflict as children may experience severe anxiety and even psychological distress, depression, low self-esteem, and sometimes substance dependence problems [START_REF] Amato | The effects of divorce and marital discord on adult children's psychological well-being[END_REF][START_REF] Cater | Childhood exposure to intimate partner violence and adult mental health problems: Relationships with gender and age of exposure[END_REF][START_REF] Davies | Isolating adult psychological correlates of witnessing parental violence: Findings from a predominantly Latina sample[END_REF][START_REF] Fergusson | Exposure to interparental violence in childhood and psychosocial adjustment in young adulthood[END_REF][START_REF] Turner | Exposure to interparental conflict and psychological disorder among young adults[END_REF]. Despite being exposed to a conflictual and even violent family climate as children, some young people and adults become resilient and do not display any symptoms [START_REF] Neighbors | Interparental conflict and relations with parents as predictors of young adult functioning[END_REF][START_REF] Suzuki | The experiences of adults exposed to intimate partner violence as children: An exploratory qualitative study of resilience and protective factors[END_REF]). This can be attributed to several protective factors that have been identified retrospectively. For example, some young adults report that safe relationships with adults on whom they could rely, a safe haven away from the interparental conflict, and out-of-school activities helped to mitigate the harmful effects of this conflict [START_REF] Gonzales | Experiencing parental violence: A qualitative examination of adult men's resilience[END_REF][START_REF] Krishnakumar | Youth perceptions of interparental conflict, ineffective parenting, and youth problem behaviors in European-American and African-American families[END_REF][START_REF] Suzuki | The experiences of adults exposed to intimate partner violence as children: An exploratory qualitative study of resilience and protective factors[END_REF].

Effect of Interparental Conflict on Parent-Child Relations

Taking the spillover hypothesis as their explanatory model, whereby disruptions in one relationship (e.g., marital) can spill over into another (e.g., parent-child), many authors have highlighted a negative influence of destructive interparental conflict on parenting and parent-child relationships [START_REF] Erel | Interrelatedness of marital relations and parent-child relations: A meta-analytic review[END_REF]Warmuth, Cummings, Mark, Davies, & Patrick, 2020). When interparental conflict becomes verbally and/or physically violent, parents may feel emotionally insecure within the couple. They may then find it hard to remain mentally available to their children and sensitive to their needs [START_REF] Coln | A unified model exploring parenting practices as mediators of marital conflict and children's adjustment[END_REF][START_REF] Davies | A process analysis of the transmission of distress from interparental conflict to parenting: Adult relationship security as an explanatory mechanism[END_REF][START_REF] Kaczynski | Marital conflict, maternal and paternal parenting, and child adjustment: A test of mediation and moderation[END_REF]. Parents subjected to stressful interparental conflict have been found to adopt stricter and more negative parenting behaviors [START_REF] Buehler | Marital conflict, ineffective parenting, and children's and adolescents' maladjustment[END_REF][START_REF] Cui | Parenting behavior as mediator and moderator of the association between marital problems and adolescent maladjustment[END_REF][START_REF] Krishnakumar | Interparental conflict and parenting behaviors: A metaanalytic review[END_REF]Krishnakumar, Buehlern, & Barber, 2003). Several young adults have also reported having conflictual relationships with their parents as children [START_REF] Miller-Graff | Parent-child warmth as a potential mediator of childhood exposure to intimate partner violence and positive adulthood functioning[END_REF][START_REF] Turner | Exposure to interparental conflict and psychological disorder among young adults[END_REF].

Links Between Interparental Conflict, Parent-Child Relations, and Symptoms Displayed by

Young Adults

To better understand the impact of destructive interparental conflict on children, research has focused on the role of parenting behaviors and, more broadly, parent-child relations. The quality of these relations has been shown to affect the adjustment of children and adolescents in a context of destructive interparental conflict. The more negative or conflictual their relations with their parents, the more disorders these young people display, and vice versa [START_REF] Bradford | When there is conflict: Interparental conflict, parent-child conflict, and youth problem behaviors[END_REF][START_REF] Buehler | Marital conflict, ineffective parenting, and children's and adolescents' maladjustment[END_REF][START_REF] Cui | Parenting behavior as mediator and moderator of the association between marital problems and adolescent maladjustment[END_REF][START_REF] Grych | Family processes that shape the impact of interparental conflict on adolescents[END_REF][START_REF] Kaczynski | Marital conflict, maternal and paternal parenting, and child adjustment: A test of mediation and moderation[END_REF][START_REF] Krishnakumar | Youth perceptions of interparental conflict, ineffective parenting, and youth problem behaviors in European-American and African-American families[END_REF]Warmuth, Cummings, Mark, Davies, & Patrick, 2020). By contrast, the long-term effects of parent-child relations on young adults who witnessed destructive interparental conflict as children have received far less attention from authors. And yet young adults' negative perceptions of the relationship they had with their parents as children can have harmful repercussions on their mental health [START_REF] Amato | The effects of divorce and marital discord on adult children's psychological well-being[END_REF][START_REF] Turner | Exposure to interparental conflict and psychological disorder among young adults[END_REF]. The impact of perceived maternal rejection on internalizing and/or externalizing problems among young adults was recently highlighted by [START_REF] Duval | Effect of perceived parent child relations on adjustment of young women exposed to mutual intimate partner violence during childhood[END_REF], while positive parent-child relations have been found to play a long-term protective role in the resilient functioning of young adults who were exposed to interparental conflict in childhood [START_REF] Miller-Graff | Parent-child warmth as a potential mediator of childhood exposure to intimate partner violence and positive adulthood functioning[END_REF].

The Present Study

Previous studies of children's experience of interparental conflict have failed to explore their degree of involvement in conflict scenes [START_REF] Kimber | The association between child exposure to intimate partner violence (IPV) and perpetration of IPV in adulthood-A systematic review[END_REF][START_REF] Knutson | Assessing children's exposure to intimate partner violence[END_REF]. And yet it is important to consider the precise nature of this conflict and of children's exposure to it if we wish to understand its subsequent impact [START_REF] Knutson | Assessing children's exposure to intimate partner violence[END_REF][START_REF] Latzman | Children's exposure to intimate partner violence: A systematic review of measurement strategies[END_REF]. In [START_REF] Fantuzzo | Children's direct exposure to types of domestic violence crime: A population-based investigation[END_REF]'s investigation into domestic violence, children reported being present in half of all episodes. More than half of them stated that they had seen and heard violent scenes between their parents. When children witness this violence first hand, it is an overwhelming sensory experience that threatens their emotional security (Goeke-Morey, [START_REF] Goeke-Morey | Children and marital conflict resolution: Implications for emotional security and adjustment[END_REF]. This insecurity may be exacerbated if their parents, who themselves are experiencing insecurity within the couple, are less emotionally available to them [START_REF] Adeyemi | Inter-parental conflict, parent-child relationship on emotional security of school-going adolescents in Ibadan South-West Local Government Area[END_REF][START_REF] Davies | Marital conflict and child adjustment: An emotional security hypothesis[END_REF][START_REF] Erel | Interrelatedness of marital relations and parent-child relations: A meta-analytic review[END_REF][START_REF] Goeke-Morey | Children and marital conflict resolution: Implications for emotional security and adjustment[END_REF][START_REF] Grych | Family processes that shape the impact of interparental conflict on adolescents[END_REF]. Paradoxically, in an attempt to recover a feeling of security, children may display negative behavioral and emotional reactions (aggressive or disruptive behaviors, crying, etc.). These reactions may in turn have repercussions on their parents' marital relations and/or their own relations with their parents [START_REF] Davies | Marital conflict and child adjustment: An emotional security hypothesis[END_REF][START_REF] Erel | Interrelatedness of marital relations and parent-child relations: A meta-analytic review[END_REF]Schermerhorn, Cho, & Cummings, 2010). If this vicious circle persists, its negative impact on wellbeing may last into young adulthood [START_REF] Davies | Marital conflict and child adjustment: An emotional security hypothesis[END_REF][START_REF] Young | Children's perceptions of parental emotional neglect and control and psychopathology[END_REF]. Studies of long-term effects mostly report negative trajectories, that is, the reproduction of violence and adjustment problems. Rather fewer retrospective studies have explored positive adjustment over the long term, which can mainly be attributed to protective factors in childhood. Several of these have emphasized the importance of good parent-child relations for subsequent resilience in young adulthood [START_REF] Amato | The effects of divorce and marital discord on adult children's psychological well-being[END_REF][START_REF] Miller-Graff | Parent-child warmth as a potential mediator of childhood exposure to intimate partner violence and positive adulthood functioning[END_REF]. To our knowledge, however, there has yet to be a retrospective study of the effects of direct exposure to destructive interparental conflict on parent-child relations and thence on the resilience of young adults.

Our research aims to investigate specifically the effects of the frequency of direct exposure (i.e., being physically close to destructive interparental conflict) on subsequent levels of resilience and perceptions of parent-child relationships. We are not seeking to find out whether some of the participants may have been abused or neglected during childhood or adolescence. From this general objective derive 3 sub-objectives: 1) Based on the recollections of the adult participants in the study, our first objective is to determine whether the frequency of direct exposure to interparental conflict in childhood influences subsequent perceptions of the quality of child-parent relationships.

We tested the following operational hypothesis:

Children's Physical Proximity to parental Conflict 8 -Individuals who were often close to interparental conflict scenes in their childhood have more negative perceptions of their past relationships with their parents than individuals who have never been close; 2) Our second objective is to assess whether this physical proximity has a long-term impact on resilience.

We tested the following operational hypothesis:

--Individuals who were often / always close to interparental conflict scenes in their childhood were more often exposed to IPV, and therefore felt a stronger feeling of insecurity than individuals who were never / rarely close; -The proximity frequency of interparental conflict scenes moderates the relationship between psychological and / or physical violence perpetrated during these scenes and the level of resilience;

3) Our third objective aims to determine whether the memories of young adults of their relationships with their parents in their childhood can explain the links between the frequency of exposure to interparental conflict, the degree of physical proximity to the scenes of conflict and the development resilient in young adults.

We tested the following operational hypothesis:

-Retrospective perceptions of parent-child relationships and the feeling of insecurity mediate the link between psychological and / or physical violence perpetrated during interparental conflicts and the level of resilience.

Children's Physical Proximity to parental Conflict 9

Method Participants, Procedure and Ethics

The study was conducted in France between 2018 and 2019. An online study was posted on social networks (Facebook, Instagram and email) and sent to students at Aix-Marseille University.

To comply with research ethics, participants, all aged between 18 and 25, read an information note explaining the research protocol before electronically signing a free and informed consent to participate. It was clearly stated that their responses were strictly anonymous. An email address was provided to answer any questions they might have about the procedure or if they needed to express themselves in case of emotional distress as a result of the questions. As the researchers were all clinical psychologists, support or counselling could be provided. At the end of the research, no such requests were identified.

The form was sent to students without distinction regarding a specific history of abuse. As a preamble we recalled that family life is often full of ups and downs, and that difficult times can be marked by more or less severe and frequent parental disputes. They were specified that our interest was specifically "in understanding what they experienced as a child (or adolescent) when they witnessed parental disputes and how they reacted".

Measures

To assess our variables, we first constructed an entry questionnaire that collected information about their age, sex, and level of study. We included the following two items, to determine the frequency of participants' physical proximity to interparental conflict and their feeling of insecurity in childhood: "When arguments broke out, you were usually [in the same room/near your parents]"

Children's Physical Proximity to parental Conflict 10 and "Generally speaking, how often did you feel unsafe at home?" Items were rated on a 5-point Likert scale: 1 (Never), 2 (Rarely), 3 (Sometimes), 4 (Often), and 5 (Always).

Second, we built a protocol from three tools. First, we took six items from the Conflict Tactics Scale 2 [START_REF] Straus | Child-report, adult-recall, and sibling versions of the Revised Conflict Tactics Scale[END_REF] to assess our participants' memories of the frequency of interparental conflict when they were children. Three items were taken from the psychological and verbal violence subscale, and one from each of the following subscales: physical violence, sexual violence, and injury.

These items were rated on a 5-point Likert scale ranging from 1 (Never) to 5 (Always). We were able to replace the words My partner in the original version with My father/mother for the purposes of our study, as the original scale can be applied very broadly, and in the past its items have been adapted to measured relations between all the different family roles, including husband-wife and parent-child [START_REF] Straus | Child-report, adult-recall, and sibling versions of the Revised Conflict Tactics Scale[END_REF]. The scale has good psychometric qualities, with a Cronbach's alpha of.79-.95 [START_REF] Straus | The revised Conflict Tactics Scales (CTS2)[END_REF].

We also used [START_REF] Wagnild | Development and psychometric evaluation of the resilience scale[END_REF]'s resilience scale, translated into French for France and Quebec by [START_REF] Ionescu | Socio-political context, risk factors and scores on Resilience scale[END_REF]. Each of its 25 items is rated on a 7point Likert scale ranging from 1 (Totally disagree) to 7 (Totally agree), with a maximum total score of 175. A score below 121 indicates a low level of resilience, a score of 121-145 a moderate level, and a score equal to or above 146 a high level [START_REF] Wagnild | The resilience scale user's guide[END_REF]. The original version has good psychometric qualities. The American version has good internal consistency, with Cronbach's alphas of .84-.94 [START_REF] Wagnild | The resilience scale user's guide[END_REF].

The Invalidating Childhood Environments Scale (ICES; [START_REF] Mountford | Development of a measure to assessing validating childhood environments in the eating disorders[END_REF] is a two-part questionnaire that retrospectively assesses the experience of living with one's parents and within a family. The first part assesses emotional experiences with each parent, via items (e.g., "When I was anxious, [my mother] ignored this" / "When I was anxious, [my father] ignored this") rated on a 5point Likert scale ranging from 1 (Never) to 5 (Always). The higher the score, the stronger the Children's Physical Proximity to parental Conflict 11 perception of an invalidating family environment. The second part assesses respondents' overall perceptions of their family style via three types (typical, perfect, and chaotic) of invalidating family environments and one validating environment [START_REF] Linehan | Cognitive-behavioral treatment of borderline personality disorders[END_REF]. In a typical family, children are supposed to control their emotions, seek success, and act like adults. In the perfect family, although everything is superficially perfect, the parents cannot stand their children expressing annoyance, anger, or fear. The priority is to hide one's emotions and make do. In the chaotic family, the parents are often physically and/or emotionally unavailable. They may engage in substance use, have mental health problems, or be in financial difficulty. Lastly, a validating family responds appropriately to children's emotions [START_REF] Mountford | Development of a measure to assessing validating childhood environments in the eating disorders[END_REF]. The items corresponding to these four family types are rated on a 5-point Likert scale ranging from 1 (Not at all like my family) to 5 (Like my family all of the time). The respondent's family may correspond to different types to varying degrees. According to Campagnone and Lo Monaco (2015), the French version has good psychometric qualities, as borne out by the internal consistency coefficient (Crombach's alpha = .83).

Data Analysis

The data were submitted to statistical processing with SPSS.24 software. As the distribution of our variables was normal, we ran parametric tests. First, we conducted descriptive analyses (means/SDs; chi 2 ). Second, we undertook means comparisons (Student's t) to highlight differences between the groups. Third and last, we tested a stepwise hierarchical multiple regression model for the resilience criterion. This allowed us to analyze the moderating effect of the physical proximity to interparental conflict variable, and the mediating effects of the perception of an invalidating family environment and feeling of insecurity variables on the relations between exposure to psychological and/or physical intimate partner violence and resilience.

Children's Physical Proximity to parental Conflict 12

Results

Sample

We recruited 963 participants with a mean age of 21.07 years (SD = 2.11). This sample comprised 847 women (Mage = 21.04 years, SD = 2.08) and 116 men (Mage = 21.27 years, SD = 2.32), 624 of whom no longer lived in the parental home. Participants' responses to the statement "When arguments broke out, you were usually [in the same room / near to your parents]" allowed us to divide them into three groups:

-Group 1 comprised 407 participants who replied "rarely or never" (52 men and 355 women; Mage = 20.96 years, SD = 2.08); -Group 2 comprised 272 participants who replied "sometimes" (29 men and 243 women; Mage = 21.1, SD = 2.17); -Group 3 comprised 284 participants who had replied "often and always" (35 men and 249 women;

Mage = 21.2, SD = 2.08).
Table 1 sets out the characteristics of our sample. It shows that the prevalence of exposure to psychological violence (at least once) in Group 3 (often/always close) during interparental conflict was 99%, compared with 95% for Group 2 (sometimes close) and 70% for Group 1 (never/rarely close) (p < .0001). As for physical and/or sexual violence, 48% of participants in Group 1 reported being exposed at least once, compared with 26.5% for Group 2, and 17% for Group 3 (p < .0001).

Analyses failed to reveal any sex-related differences.

Insert Table 1 about here

Children's Physical Proximity to parental Conflict 13

Intergroup Comparison: Analyses of Variance

Analyses of variance (ANOVAs) allowed us to test our first and second hypotheses whereby individuals who were more frequently close to interparental conflict as children (Group 1) have a stronger perception of an invalidating family environment and recall a greater feeling of insecurity than those in the other groups, and were more exposed to intimate partner violence.

We observed that the closer participants had physically been to interparental conflict as children, the stronger their perception of an invalidating family environment and the greater their feeling of insecurity and exposure to violence. Individuals who reported being often/always close scored higher on the ICES than those in the other groups (MGroup1 = 64.57, SD = 18.54 / MGroup2 = 69.99, SD = 18.01 / MGroup3 = 77.84, SD = 19.83; p < .0001). Chaotic, typical and perfect (i.e., invalidating) family types were more common among participants who were often/always close (p < .0001), whereas the validating family type was more common among participants who were never/rarely close (F = 32.51, p < .0001). Participants who were often/always close reported a significantly higher level of exposure to interparental conflict that those who were sometimes or rarely/never close (MGroup1 = 15.73, SD = 4.06 / MGroup2 = 16.82, SD = 3.52 / MGroup3 = 18.52, SD = 3.97; p < .0001), especially when the violence was psychological (MGroup1 = 10.65, SD = 3.46 / MGroup2 = 11.91, SD = 3.14 / MGroup3 = 13.33, SD = 3.43; p < .0001). The feeling of insecurity during interparental conflict was far greater in Group 1 (M = 2.4, SD = 1.07; p < .0001) than in Groups 2 (M = 1.79, SD = .96; p < .0001) or 3 (M = 1.5, SD = .82; p < .0001).

Insert Table 2 about here

Children's Physical Proximity to parental Conflict 14

Moderating Effect

We tested a third hypothesis whereby the frequency of physical proximity to interparental conflict has a moderating effect on the link between exposure to intimate partner violence and the subsequent resilience of the exposed child.

According to [START_REF] Baron | The moderator-mediator variable distinction in social psychological research: Conceptual, strategic, and statistical considerations[END_REF], to measure a moderating effect, a new variable representing the product of the two predictors must be constructed, in order to carry out a stepwise hierarchical multiple regression. We expected our significant interaction variable (Violence x Frequency of proximity) to demonstrate a moderating effect of proximity on the link between violence and resilience. However, our results failed to reveal an interaction effect, indicating that frequency of proximity did not play the expected role of moderating variable.

By contrast, Table 3, which summarizes the regression analyses, shows a moderating effect of frequency of proximity to interparental conflict on the perception of an invalidating family environment. The latter was significantly and positively influenced by proximity (β = .106, p < .001), such that the closer participants were to interparental conflict as children, the stronger their perception of an invalidating family environment.

Another method that can be used to highlight the moderating effect of a variable involves comparing predictor-criterion correlations between groups with a high or low score on the moderator (Racle & Irachabal, 2001). We therefore carried out a regression on the three proximity groups.

Insert Table 3 about here

Results indicated an increase in R 2 , indicating that greater exposure to violence had a more negative effect on resilience in Group 1 (R 2 = .049, p < .0001) than in Group 2 (R 2 = .011, p < .03).

Children's Physical Proximity to parental Conflict 15

Mediating Effect

Table 4 summarizes the regression analyses that allowed us to test our final hypothesis on the mediating effects of perception of an invalidating family environment and feeling of insecurity on the link between the predictors (exposure to violence and frequency of proximity) and resilience.

We observed a mediating effect of the perception of an invalidating family environment variable, but no significant mediating effect of feeling of insecurity. These analyses met the required conditions to detect a mediation effect. Results highlighted a significant increase in R 2 when the model included perception of an invalidating family environment (R 2 = .010 and R 2 = .060; p < .0001), with a reduction in the predictive power of the exposure to violence variable from β = -.112 to β = -.06 (p < .02), and a significant increase in R 2 when we entered the proximity variable. The two predictive variables significantly and positively influenced perception of an invalidating family environment (R 2 = .234, p < .001). However, exposure to violence (β = .427, p < .0001) seemed a more discriminating variable than frequency of proximity (β = .108, p < .001). Exposure to violence had a greater influence on perception of an invalidating family environment than proximity did. In summary, our data revealed that violence and proximity negatively influenced perception of an invalidating family environment, thereby making young adults less resilient.

Insert Table 4 about here

Discussion

Our main research objective was to retrospectively ascertain whether young adults' memories of their relations with their parents as children can explain the links between the frequency of exposure to interparental conflict, physical proximity to conflict scenes, and resilient development.

In line with previous retrospective studies, our sample of young adults had been exposed to destructive interparental conflict in their childhood and/or adolescence. This conflict was mostly Children's Physical Proximity to parental Conflict 16 characterized by verbal or psychological violence [START_REF] Cater | Childhood exposure to intimate partner violence and adult mental health problems: Relationships with gender and age of exposure[END_REF][START_REF] Fergusson | Exposure to interparental violence in childhood and psychosocial adjustment in young adulthood[END_REF]. Virtually all (99%) had been exposed at least once to psychological violence, and nearly half (48%) to physical/sexual violence. Our data showed that the risk of witnessing physical violence was higher among young adults who had often/always been close to scenes of conflict (Group 1) than among those who had more often remained at a distance. This observation echoed the study by [START_REF] Papp | Marital conflicts in the home when children are present versus absent[END_REF], who showed that interparental conflict is more destructive when a child is present, as the parents are less liable to engage in constructive conflict tactics. In all probability, their emotional distress is exacerbated by the child's presence and possibly even by his or her reactions.

Only young adults who had sometimes been close to interparental conflict had a low level of resilience, when we applied Wagnild ( 2009)'s cut-off score. However, when we compared our results with those of Jourdan- Ionescu et al. (2015), we found that our entire sample had a lower overall level of resilience. This result is in line with other studies showing that young adults exposed to destructive interparental conflict in childhood may develop psychological and even psychiatric disorders [START_REF] Cater | Childhood exposure to intimate partner violence and adult mental health problems: Relationships with gender and age of exposure[END_REF][START_REF] Turner | Exposure to interparental conflict and psychological disorder among young adults[END_REF]. Jourdan- Ionescu and colleagues (2015) administered Wagnild and Young's Resilience Scale to 162 students to measure its internal consistency. Female respondents had a mean score of 133.88, and male respondents a mean score of 140.41. As for our sample, mean scores on this same scale were 124.75 for Group 1, 120.75 for Group 2, and 122.25 for Group 3. According to the cut-offs established by the scale's authors, the mean scores of Groups 1 and 3 were within the mean for resilience scores, whereas the mean score of participants in Group 2 indicated a low level of resilience. Overall, the participants in our sample had a lower level of resilience than those in the Quebec study (Jourdan-Ionescu et al., 2015).

We also found, based on the study by Campagnone and Lo Monaco (2015), that the young adults in our sample had a strong perception of an invalidating family environment, especially those Children's Physical Proximity to parental Conflict 17 who had frequently been close to interparental conflict as children. In the study validating the Frenchlanguage version of the ICES (Campagnone & Lo Monaco, 2015), authors administered the questionnaire to 585 students. With reference to this study, we found that participants who were exposed to occasional psychological and/or physical intimate partner violence and those who were physically close to scenes of interparental conflict had stronger perceptions of an invalidating family environment. Participants who had never been exposed or who had never been close to such conflict had scores within the mean.

In this sense, our analyses validated our first hypothesis, whereby individuals who found themselves in close proximity to interparental conflict as children have more negative retrospective perceptions of their child-parent relations than those who rarely if ever witnessed destructive interparental conflict. Our results are in line with those of other retrospective studies among young people and adults who were exposed to interparental conflict as children [START_REF] O'brien | Lifting the domestic violence cloak of silence: Resilient Australian women's reflected memories of their childhood experiences of witnessing domestic violence[END_REF][START_REF] Turner | Exposure to interparental conflict and psychological disorder among young adults[END_REF]. The young adults in our study had memories of negative parenting, in the sense that they did not recall their parents responding to their emotional needs in an appropriate manner when they were children and/or adolescents. [START_REF] Davies | A process analysis of the transmission of distress from interparental conflict to parenting: Adult relationship security as an explanatory mechanism[END_REF] observed that destructive interparental conflict at Time 1 in their longitudinal study was predictive of a reduction in the parents' emotional availability 12 months later. Owing to the emotional distress they experience as a result of this conflict, parents may adopt poor childrearing practices reflecting an inability to be receptive to their children's needs and emotions (Campagnone & Lo Monaco, 2015).

Results validated our second hypothesis, whereby individuals who are often physically close to interparental conflict as children are also more exposed to violence, and therefore experience a greater feeling of insecurity. We found that the closer our participants had been to interparental conflict, the greater their exposure to violence at the time, and the greater their feeling of insecurity.

Children's Physical Proximity to parental Conflict 18

There are two possible interpretations of this finding. First, a child's physical proximity to interparental conflict tends to exacerbate it. This interpretation is supported by the conclusions of a national French statistical survey [START_REF] Brown | La place de l'enfant dans les conflits et les violences conjugales[END_REF], which emphasized that having children heightens the risk of tension within a couple, as there are more sources of potential conflict, such as childrearing. In particular, when children are present, there are more arguments about them. Parents therefore experience greater emotional distress than they would do in the absence of children, thus exacerbating the conflict [START_REF] Papp | Marital conflicts in the home when children are present versus absent[END_REF]. Second, when interparental conflict becomes more violent and more frequent, children are more likely to be close, and thus to feel unsafe. Mothers report that the more longstanding the intimate partner violence, the more their children witness it [START_REF] Brown | La place de l'enfant dans les conflits et les violences conjugales[END_REF]. [START_REF] Grych | Family processes that shape the impact of interparental conflict on adolescents[END_REF] also showed that the more arguments parents have, the greater their children's voluntary or involuntary involvement, attesting to their presence during these events. Frequent violent interparental conflict is the reflection of a dysfunctional couple, and is often the sign of a dysfunctional family. We can assume that children are then at greater risk of being set upon by one or other parent during a conflict. In an attempt to regain their emotional security, children may also intervene in violent scenes between their parents [START_REF] Davies | Marital conflict and child adjustment: An emotional security hypothesis[END_REF].

Analyses conducted according to [START_REF] Baron | The moderator-mediator variable distinction in social psychological research: Conceptual, strategic, and statistical considerations[END_REF]'s statistical method also allowed us to validate our third hypothesis, but only partially so, as frequency of proximity did not moderate the link between destructive interparental conflict and resilience. We nevertheless observed a moderating effect of frequency of proximity on the link between intimate partner violence and perception of an invalidating family environment. In addition, the more often our young adults had been close to interparental conflict as children, the more negative their retrospective perception of parent-child relations. These moderating effects of frequency of proximity can be explained by the consequences of children being present during interparental conflict. Frequent physical proximity suggests that the young adults were very much involved in violent scenes when they were children and/or adolescents. They may have chosen to be present out of a desire to understand what was happening, to stop the conflict or at least calm the two sides down, intervene if necessary, take the side of one of the parents, or protect a parent or sibling. Then again, they may have been unwittingly drawn into the conflict quite simply because of the cramped living space. [START_REF] Fosco | Adolescent triangulation into parental conflicts: Longitudinal implications for appraisals and adolescent-parent relations[END_REF] showed that the more the presence of children or adolescents is an issue for parents, the more negative parent-child relations are likely to be. They also found that if the boundary between the various family subsystems is breached, the feelings of trust and security that children/adolescents have toward their parents are likely to be negatively affected [START_REF] Fosco | Adolescent triangulation into parental conflicts: Longitudinal implications for appraisals and adolescent-parent relations[END_REF], as they no longer perceive their parents to be sources of support.

Results only partially validated our fourth and final hypothesis, for contrary to the descriptions in the literature, we failed to observe a mediating effect of feeling of security on the link between destructive interparental conflict and resilience. Even if the young adults in our sample had felt unsafe as children or adolescents during scenes of interparental conflict, this experience did not massively lower their resilience, as levels remained around the mean for most of them. Our results were not, therefore, in line with research suggesting that a high level of threat/insecurity can harm the psychological health of children and adolescents, notably by hindering their resilient development [START_REF] Davies | Marital conflict and child adjustment: An emotional security hypothesis[END_REF][START_REF] Fosco | Making sense of family violence: Implications of children's appraisals of interparental aggression for their short-and longterm functioning[END_REF]. Our results indicate that even when there is frequent exposure to destructive interparental conflict, the harmful effects of the resulting insecurity recede and may even disappear completely over time. They can probably be explained by the fact that two thirds (n = 624) of the young adults in our sample were no longer living under their parents' roof and were therefore no longer (or less) exposed to destructive interparental conflict. [START_REF] El-Sheikh | Dynamic patterns of marital conflict: Relations to trajectories of adolescent adjustment[END_REF] showed that young people exposed to low and diminishing levels of destructive interparental conflict have fewer symptoms than those experiencing greater exposure. Moreover, by the time they reach adulthood, young people are more psychologically mature, and can therefore give a meaning to what they experienced in childhood, and Children's Physical Proximity to parental Conflict 20 distance themselves from it. Protective factors (individual, family and friends, community), such as the ability to project oneself into a pleasant future, good cognitive skills, and a good social support network, may also reduce the feeling of insecurity over time.

As the destructive interparental conflict involved only minor, occasional violence, we can further assume that the feeling of insecurity experienced during scenes of conflict was purely intermittent, and not sufficiently intense to trigger the resilience process. In order to withstand adversity, we first have to be exposed to it. This notion is illustrated by two studies where Wagnild and Young (1993)'s scale was used to measure students' resilience. Jourdan- Ionescu et al. (2015) found that the two students in their sample (N = 162) with the highest resilience scores had a history of child abuse, while in their study of resilience among students in France and three African countries, [START_REF] Ionescu | Socio-political context, risk factors and scores on Resilience scale[END_REF] found that Algerian students, characterized by moderately high risk factors, had the highest mean resilience scores. By contrast, French students, who benefitted from protective factors in the form of social support and few risk factors, had the lowest resilience scores.

Lastly, perception of an invalidating family environment mediated the link between frequency of proximity to conflict, exposure to destructive interparental conflict, and resilience. This was consistent with longitudinal and retrospective studies showing that the effects of interparental conflict on young adults' psychopathological symptoms are mediated by parenting behaviors or parent-child relations [START_REF] Amato | The effects of divorce and marital discord on adult children's psychological well-being[END_REF][START_REF] Cui | Parenting behavior as mediator and moderator of the association between marital problems and adolescent maladjustment[END_REF][START_REF] Miller-Graff | Parent-child warmth as a potential mediator of childhood exposure to intimate partner violence and positive adulthood functioning[END_REF]. In contrast to previous studies, however, our results highlighted the effect of physical proximity. They also showed that intensity of conflict influences the negative retrospective perception of parent-child relationships more than the frequency of proximity. In other words, frequent physical proximity to scenes of destructive interparental conflict has a negative impact on young adults' perceptions of these relations. These negative perceptions, reflecting the experience of emotional neglect, can affect young adults' resilient development. This means that perceived Children's Physical Proximity to parental Conflict 21 emotional support from parents during childhood is a long-term protective factor against the harmful effects of physical proximity to violent interparental conflict.

Several methodological limitations arising from our choice of tools and the way we recruited our sample nevertheless restrict how far our results can be generalized. First, our sample of young adults was entirely made up of students-mainly women-, thus limiting the scope of our conclusions.

Analyses based on an unselected sample are therefore needed to confirm and complete our results.

Second, the questionnaire we used to measure exposure to interparental conflict did not allow us to assess the potentially chronic nature of the violence. We do not know exactly how old our participants were when they witnessed scenes of destructive interparental conflict. Nor do we know whether they considered this exposure to be in the distant past, more recent, or recurrent. Third, it is possible that some of the participants have experienced sexual abuse, which may impact level of resilience, sense of security and perceptions of relationships with parents. This aspect will need to be investigated in a future study. Fourth the responses to the questionnaire on the childhood family environment may have been influenced by the status of respondents' current relations with their parents. Tensions in parent-child relations resulting from interparental conflict may either persist [START_REF] Turner | Exposure to interparental conflict and psychological disorder among young adults[END_REF] or subside over time. Fifth, given that ours was a retrospective study, it would also be unwise to establish any causal links between our variables. However, our study could shed light on professional practice on the issue of prevention with families and parental guidance towards positive parenting. Our findings encourage reflection on the importance of protecting children from inter-parental conflict. They promote awareness of the negative effects of interparental conflicts on young adults. The challenge of supporting them is important because most of them will become parents and risk reproducing the same conflictual attitudes.

Children's Physical Proximity to parental Conflict 22

Conclusion

There has been scant research on the physical proximity of children to scenes of interparental conflict, as reported retrospectively by young adults. Most of the available data on children's absence or presence during arguments come from the accounts of parents, mainly mothers. In this regard, our study provides a new perspective on the topic of children who witness interparental conflict, some of it violent. Our approach yielded two main results. First, the frequency of this proximity is a major long-term risk factor in terms of perceived parent-child relations and child development. Second, the perception of supportive parent-child relations can be a long-term protective factor against the effects of destructive interparental conflict on resilience. These two results should inform the debate about how best to support families characterized by violent transactions, at all levels of prevention. Our approach also opens up new avenues for research, notably the retrospective assessment of children's degree of involvement in interparental conflict, focusing on their affective and behavioral reactions.

There are many issues arising from the presence of children during interparental conflict, both for the children themselves and for their parents. This research would therefore be worthwhile for three reasons. First, it would provide a means of studying the effects of children's presence, depending on whether it is voluntary or involuntary. Second, it would help us to understand what prompts children to intervene (or refrain from intervening) in interparental violence. Third, it would enable us to assess the long-term effects of these reactions on children's resilience.

  Children's Physical Proximity to parental Conflict 5

  Children's Physical Proximity to parental Conflict 19


Fanny Vogelweith
email: fannyvogelweith@gmail.com

Yannick Moret

Karine Monceau

Denis Thiéry

Jérôme Moreau

Denis Thiery

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet

Keywords: ecological immunity, Eupoecilia ambiguella, grape varieties, hemocyte differentiation, Lepidoptera, Tortricidae

come

The relative abundance of hemocyte types in a

polyphagous moth larva depends on diet.

Introduction

In insect immunity, core processes such as coagulation, nodulation, phagocytosis and encapsulation are strongly associated with hemocytes [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF]. They recognize and destroy invading pathogens and apoptotic cells during phagocytosis, and aggregate large numbers of bacteria during the nodulation process. They also bind to larger targets including eggs of parasitoids, nematodes and protozoa by forming a multilayer capsule around the intruder (so-called encapsulation; [START_REF] Marmaras | Regulators and signalling in insect haemocyte immunity[END_REF].

Hemocytes circulate freely in the hemocoel, and are produced either by hematopoietic organs or through cell division [START_REF] Gillespie | Biological mediators of insect immunity[END_REF][START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF][START_REF] Strand | The insect cellular immune response[END_REF][START_REF] Tan | Characterization of hemocytes proliferation in larval silkworm, Bombyx mori[END_REF]. They differentiate into cell types that have various functions in pathogen defense [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF], and have been classified according to their morphology, function and certain molecular biomarkers. In Lepidopteran caterpillars, including Bombyx mori, five types of hemocytes have been described [START_REF] Ling | Hemocyte differentiation in the hematopoietic organs of the silkworm, Bombyx mori: prohemocytes have the function of phagocytosis[END_REF][START_REF] Strand | The insect cellular immune response[END_REF][START_REF] Tan | Characterization of hemocytes proliferation in larval silkworm, Bombyx mori[END_REF]: prohemocytes, granulocytes, plasmatocytes, spherulocytes and oenocytoids. Prohemocytes are the precursor of the other types; they are produced and reside primarily in hematopoietic organs, and thus rarely circulate in the hemocoel [START_REF] Lanot | Postembryonic hematopoiesis in Drosophila[END_REF]. Granulocytes (GR) are usually the most abundant type in the hemolymph; they adhere strongly to the surface of foreign bodies, function as phagocytes, and are involved in encapsulation [START_REF] Strand | The insect cellular immune response[END_REF].

Plasmatocytes (PL) are polymorphic in shape; they spread asymmetrically on foreign surfaces, and participate in capsule-formation during the encapsulation reaction [START_REF] Strand | The insect cellular immune response[END_REF]. In contrast, spherulocytes (SP) are non-adhesive hemocytes and their role in insect immunity is still unknown, although it has been suggested that they are involved in the transport of cuticular components [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF]. Oenocytoids (OE) are nonadhesive hemocytes that contain the main components of the phenoloxidase (PO) cascade [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF]. The proportion of each hemocyte type can vary according to numerous factors including nutrition.

Nutrition is now recognized as a critical factor in immune defense and resistance to pathogens [START_REF] Lazzaro | Immunity in a variable world[END_REF][START_REF] Ponton | Nutritional Immunology: A Multi-Dimensional Approach[END_REF]Vogelweith et al., 2013a).

Experimental studies of insects have demonstrated that food deprivation affects immune responsiveness [START_REF] Ayres | The Role of Anorexia in Resistance and Tolerance to Infections in Drosophila[END_REF][START_REF] Kapari | Defoliating insect immune defense interacts with induced plant defense during a population outbreak[END_REF][START_REF] Siva-Jothy | Short-term nutrient deprivation affects immune function[END_REF][START_REF] Yang | Effects of host-plant shift on immune and other key life-history traits of an eruptive Geometrid, Epirrita autumnata (Borkhausen)[END_REF] and changes in the expression of several immunity genes [START_REF] Pletcher | Genome-wide transcript profiles in aging and calorically restricted Drosophila melanogaster[END_REF]. Immune effectors can also be affected by changes in food composition [START_REF] Babin | Dietary Supplementation with Carotenoids Improves Immunity without Increasing Its Cost in a Crustacean[END_REF][START_REF] Cotter | Macronutrient balance mediates trade-offs between immune function and life history traits[END_REF][START_REF] Povey | Can the protein costs of bacterial resistance be offset by altered feeding behaviour[END_REF][START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF]. For example, [START_REF] Shikano | Indirect plant-mediated effects on insect immunity and disease resistance in a tritrophic system[END_REF] showed that Trichoplusia ni larvae reared on broccoli had more hemocytes than those reared on cucumber. Surprisingly, the influence of diet on the concentration of the different hemocyte types remains unclear. This issue is relevant since hemocyte types have different roles in immunity, and nutrition-dependent variation in their relative abundance might thus affect the immune response of phytophagous larvae. Indeed, [START_REF] Klowden | Physiological systems in insects[END_REF] suggested that GR are also involved in nutrient transport. Thus, it could be hypothesized that food deprivation or poor quality food induces a nutritive stress, reducing the proportion of GR hemocytes and affecting the encapsulation process.

Tortricid moths, including Lobesia botrana and Eupoecilia ambiguella, are the most harmful pests of grapes in Europe and North America. The larvae are polyphagous, and can develop on almost any grape variety and more than 25 other host plants [START_REF] Thiery | Relative performance of European grapevine moth (Lobesia botrana) on grapes and other hosts[END_REF]. Natural populations of tortricid moths are the target of numerous natural enemies and face variable changes in attacks by natural enemies, in both time and space [START_REF] Moreau | Species diversity of larval parasitoids of the European grapevine moth (Lobesia botrana, Lepidoptera: Tortricidae): The influence of region and cultivar[END_REF]. For example, biological control methods use both entomopathogenic microorganisms and parasitoids to control moth populations. However, the success of the attacks depends on the ability of the pest to defend itself mainly via its immune system. Particularly, hemocytes have been shown to be an important parameter of the insect immune system because of their involvement in encapsulation [START_REF] Eslin | Hemocyte load and immune resistance to Asobara tabida are correlated in species of the Drosophila melanogaster subgroup[END_REF][START_REF] Carton | Insect immune resistance to parasitoids[END_REF]. However, not all hemocyte types have the same role in immunity [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF] and not all types fight against the same enemies. E. ambiguella can grow on different host plants that affect its immune parameters [START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF]. For this reason, we considered it important to characterize the hemocyte types and investigate how their relative abundance varies among host plants to measure the effect of host plant on hemocyte type. This would be important if larvae can display differential resistance to natural enemies depending on their relative investment in the proportion of each hemocyte type.

In this study, we characterized the different types of hemocytes in E. ambiguella larvae, and assessed the influence of various artificial diets enriched with different grape varieties by measuring the concentration/abundance of each hemocyte type as a function of the food ingested by the larva. Because hemocytes are also expected to vary in numbers and proportion upon infection, we quantified changes in their concentration in response to a standard immune challenge mimicking a bacterial infection.

Materials and methods

a) Insect rearing

The insects used in this study came from an inbred stock of the European grape berry moth, E. ambiguella (Lepidoptera, Tortricidae) reared at the INRA of Bordeaux (Aquitaine, France) for several years. This stock is based on a great number of caged adults (several thousand per week), to which wild adults are regularly added. We found a very similar pattern in terms of the basal immunity level and parasitoid escape behavior between the inbred stock and wild lines sampled in French vineyards [START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF]. Thus, the results obtained for this strain are likely to be relevant to field populations. Larvae were maintained in boxes (18  11.5  7cm) on a semi-artificial diet (described in [START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF] at a density of ca. 100 individuals per 300 ml of diet.

b) Artificial diets

Four experimental diets were prepared following the method described by [START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF]. Specifically, a rearing diet without added berries was used as the control, and three test diets were prepared using berries from a different grape variety, respectively: 'Chardonnay', 'Chasselas' and 'Gewürztraminer'. During the last week of July 2009, insecticide-free bunches of berries for the diets were collected at the pre-veraison stage from the gene collection of grape plants 'Domaine de la Grande Ferrade', INRA-Bordeaux Aquitaine; this stage corresponds to the grape phenology on which the second annual generation of E. ambiguella occurs.

Newly hatched larvae (< 24 h) were individually reared in centrifuge tubes containing 1.5 ml of diet, which is sufficient for the larvae to complete development (Moreau et al., 2006a;Moreau et al., 2006b;[START_REF] Thiery | Relative performance of European grapevine moth (Lobesia botrana) on grapes and other hosts[END_REF]. The lids of the tubes were pierced with a needle to allow air circulation. Larvae were maintained until the 5 th larval instar stage under standard laboratory conditions (22 ± 1°C, 70 ± 10% r.h., photoperiod: L16:D8).

c) Collection of hemolymph and immune challenge

Hemocytes were extracted from 5 th instar larvae using the method described by [START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF]2013a). Briefly, the larvae were anesthetized on ice for 20 min, and 1 µl of hemolymph was then collected and flushed into a micro-centrifuge tube containing 20 µl of sodium cacodylate/CaCl2 buffer (0.01M sodium cacodylate, 0.005M CaCl2; pH 6.5). This sample was used to measure the total concentration of hemocytes, and to characterize the different hemocyte types as well as their relative concentration (see section d. and results).

Following this first hemolymph collection, larvae were immune challenged in the posterior part of the ventral side of the abdomen with a sterile needle dipped in a concentrated suspension of heat-killed Arthrobacter globiformis (ca. 10 9 cells ml -1 ). This bacterium is commonly used in the protocol testing of antimicrobial activity [START_REF] Sadd | Facultative but persistent transgenerational immunity via the mother's eggs in bumblebees[END_REF][START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF]Vogelweith et al., , 2013a[START_REF] Vogelweith | Food-mediated modulation of immunity in a phytophagous insect: an effect of nutrition rather than parasitic contamination[END_REF], as it is very sensitive to antimicrobial peptides of insects [START_REF] Dubuffet | Transgenerational Immune Priming Protects the Eggs Only against Gram-Positive Bacteria in the Mealworm Beetle[END_REF]. After the immune challenge, larvae were kept individually in micro-centrifuge tubes for 24 h under standard conditions before a second sample of hemolymph was collected [START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF](Vogelweith et al., , 2013b[START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF]. The delay between collecting the two hemolymph samples was determined based on preliminary experiments showing that the concentration of hemocytes stabilizes 24 hours after the immune challenge at the earliest (Supplementary material 1). This second sample of hemolymph allowed the concentration of each hemocyte type and the total concentration of hemocytes after an immune challenge to be measured. All hemolymph samples were assessed immediately to avoid coagulation and desiccation of the hemocytes.

We noted that the immune challenge induced mortality (5-10%) equally distributed among diets. We tested 23 larvae reared on Chardonnay, 23 on Chasselas, 28 on Gewürztraminer and 22 on the Control diet.

d) Hemocyte concentration and characterization

Hemocyte concentration was estimated using an improved Neubauer hemocytometer counting chamber and phase contrast microscopy (magnification 400×). Every sample of hemolymph was counted twice, once for the total number of hemocytes and once for the characterization and number of each hemocyte type.

Hemocyte characterization was performed using the hemolymph sample that had been taken before the immune challenge. Hemocyte types were characterized based on their previously reported morphological differences [START_REF] Beaulaton | Hemocytes and hemocytopoiesis in silkworms[END_REF][START_REF] Falleiros | Ultrastructural and quantitative studies of hemocytes in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Pyralidae)[END_REF][START_REF] Ribeiro | Insect haemocytes: What type of cell is that?[END_REF]. Accordingly, we considered the cell shape (round, oval, deformed) and size (big, small), the size of the nucleus and its position in the cell (in the middle or offset of the cell), and the cytoplasm compounds (presence/absence of granules and spherules, transparent or opaque).

e) Larval body size

Following the extraction of the first hemolymph sample, larval body size was estimated by measuring the maximum head capsule width (HC width; [START_REF] Delbac | Larval instars determination for the European Grapevine Moth (Lepidoptera: Tortricidae) based on the frequency distribution of head capsule widths[END_REF] using a Nikon SMZ-10A stereoscopic microscope and a VTO 232 video analysis system (Linkam Scientific Instruments). The HC width is the most reliable measure of body size in most lepidopteran larvae [START_REF] Delbac | Larval instars determination for the European Grapevine Moth (Lepidoptera: Tortricidae) based on the frequency distribution of head capsule widths[END_REF][START_REF] Godin | Head capsule width as an instar indicator for larvae of thecranberr y fruitworm (Lepidoptera: Pyralidae) in southeastern New Brunswick[END_REF][START_REF] Panzavolta | Instar determination for Pissodes castaneus (Coleoptera: Curculionidae) using head capsule widths and lengths[END_REF]Vogelweith et al., 2013b).

f) Data analysis

The effect of diets on the total number of hemocytes and on the concentration of each hemocyte type before the immune challenge was tested using Kruskal-Wallis tests because the data did not meet the assumptions of normality and homoscedasticity for parametric tests (tested using with Shapiro-Wilk and Levene tests respectively). Kruskal-Wallis multiple comparisons post hoc tests (MC) were used when needed to test differences among diets. The effect of both diet and immune challenge on the total concentration of hemocytes and the concentration of each hemocyte type (arcsine square root transformed) were analyzed using a linear mixed effects model that included individual identity as a random factor within the diet effect. The statistical significance of the effects of diet and immune challenge were tested using F-ratio statistics. When the data did not meet the conditions of normality and homoscedasticity, linear mixed effect models based on rank transformation were used instead of the classical non-parametric Friedman tests [START_REF] Baguley | Serious stats: A guide to advanced statistics for the behavioral sciences[END_REF].

Our initial data analyses revealed that larval body size was neither related to the total concentration of hemocytes nor to the concentration of single hemocyte types, irrespective of the time of measurement (i.e. before or after exposure). We hence removed this variable from all further analyses. Statistical analyses were performed using the R software (version 3.1.1; R Development Core Team, 2008) complemented with the lawstat (Levene test) and nlme (linear mixed effects model) packages.

Results

a) Hemocyte types and morphology

Fifth instar larvae of E. ambiguella contained four types of hemocytes that were easily differentiated: granulocytes (GR), plasmatocytes (PL), oenocytoids (OE) and spherulocytes (SP) (Fig. 1). The presence of granules makes GR easy to differentiate from the other types; the GR cells appear as round cells with a small and round nucleus (Fig. 1a,e). PL were also round cells of variable size (Fig. 1c,f), in which the nucleus was either round or bi-lobed, and was relatively large in the cell. SP were also easy to recognize because of their inclusions and spherules, which occupied most of the cytoplasm and deformed the cell and the nucleus (Fig. 1d,h). OE had a dense and opaque cytoplasm with a small overhanging nucleus (Fig. 1c,g).

b) Diet effect on the total concentration of hemocytes before the immune challenge

The total concentration of hemocytes was influenced by the larval diet (Wilcoxon text, χ ²3 = 11.08; p = 0.01) (Fig. 2, before challenge). Larvae reared on Gewürztraminer had more hemocytes than larvae reared on the control diet. There was no difference between larvae reared on Chardonnay and Chasselas, or between any other combinations of diets (Fig. 2, before immune challenge).

c) Diet effect on each hemocytes type before the immune challenge

Irrespective of the diet, GR were the most abundant cells in the hemolymph with an average number of 13603 hemocytes/µl (95% CI = [11906; 15300], accounting for 67% of the hemocytes (Fig. 3, before immune challenge). PL were the second most abundant hemocytes with an average number of 5955 hemocytes/µl (95% CI = [5115; 6795], accounting for 29%. SP (mean = 518 hemocytes/µl; 95% CI = [384; 652]) and OE (mean = 136 hemocytes/µl ; 95% CI = [102; 170]) were much less abundant, accounting for 3% and 1% of the hemocytes, respectively.

Larval diet affected the concentrations of GR (χ²3 = 12.83; p = 0.005), PL (χ²3 = 8.86; p = 0.03) and SP (χ²3 = 23.64; p < 0.0001) but not that of OE (χ²3 = 1.05; p = 0.79) (Fig. 3, before immune challenge). The concentration of GR was lower in larvae reared on the control diet than in those reared on the Gewürztraminer and Chardonnay diets (Fig. 3a, before immune challenge). Larvae reared on the control diet also had less PL than those reared on Gewürztraminer (Fig. 3b, before immune challenge). Except for larvae reared on Gewürztraminer and control diets which did not differ, all larvae reared on the other diets had different concentration of SP (Fig. 3c, before immune challenge).

d) Diet and immune challenge effect on the total concentration of hemocytes and on the concentration of each hemocyte types

The total concentration of hemocytes after the immune challenge was only affected by that challenge (F1,110 = 18.44 ; p < 0.0001), but not by the larval diet (F3,74 = 1.62; p = 0.19) or an interaction between challenge and diet (F3,110 = 2.62 ; p = 0.06). Irrespective of the diet, the total concentration of hemocytes increased after the immune challenge (Fig. 2, before and after immune challenge).

The immune challenge affected the concentration of all hemocyte types, either alone, or in an interaction with the diet (Table 1; Fig. 3). Specifically, the concentration of GR in the hemolymph of larvae increased irrespective of the diet after the immune challenge (MC: p < 0.0001; Fig. 3a). In contrast, the changes in the concentration of PL, SP and OE caused by the immune challenge depended on the diet (Table 1; Fig. 3b,c,d). The concentration of PL decreased after the immune challenge in larvae reared on Gewürztraminer (MC: p = 0.01) and increased in larvae reared on the control diet (MC: p = 0.04) (Fig. 3b). The concentration of PL did, however, not change in larvae reared on Chardonnay and Chasselas (MC: p = 0.96 and p = 0.73, respectively). The concentration of OE and SP increased after the immune challenge in larvae reared on the control diet (MC, OE: p = 0.006; SP: p = 0.0006; Fig 3c,d), but did not change in larvae reared on Chasselas, Chardonnay or Gewürztraminer (MC, OE, Chardonnay: p = 0.16; Chasselas: p = 0.85; Gewürztraminer: p = 0.79 and SP, Chardonnay: p = 0.79; Chasselas: p = 0.43; Gewürztraminer: p = 0.79, Fig. 3c,d).

Discussion

This study aimed to characterize the different types of hemocyte in 5 th instar larvae of E. ambiguella, and to test the effects of diet and immune challenge on the concentration of each hemocyte type. We showed that the total concentration of hemocytes and the concentration of the four hemocyte types varied among diets and in response to the immune challenge.

Consistent with other studies in lepidopteran species [START_REF] Falleiros | Ultrastructural and quantitative studies of hemocytes in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Pyralidae)[END_REF][START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF][START_REF] Ling | Hemocyte differentiation in the hematopoietic organs of the silkworm, Bombyx mori: prohemocytes have the function of phagocytosis[END_REF][START_REF] Ribeiro | Insect haemocytes: What type of cell is that?[END_REF], we identified granulocytes, oenocytoids, plasmatocytes and spherulocytes hemocytes based on their morphological features. However, we did not detect prohemocytes (PR), although these have been reported in larvae of other lepidopteran species [START_REF] Falleiros | Ultrastructural and quantitative studies of hemocytes in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Pyralidae)[END_REF]. We tested only mature larvae at the end of the larval stage. [START_REF] Falleiros | Ultrastructural and quantitative studies of hemocytes in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Pyralidae)[END_REF] reported that the proportion of PR decreases with larval age until it reaches zero, which may explain why we found no PR in 5 th instar larvae of E. ambiguella.

As in other species of Lepidoptera, including Diatraea saccharalis [START_REF] Falleiros | Ultrastructural and quantitative studies of hemocytes in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Pyralidae)[END_REF], Euclea delphinii and Lithacodes fasciola [START_REF] Stoepler | Hemocyte Density Increases with Developmental Stage in an Immune-Challenged Forest Caterpillar[END_REF], GR and PL were the most abundant circulating hemocytes in E. ambiguella. GR are involved in the recognition of foreign bodies, phagocytosis, encapsulation and nodulation [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF][START_REF] Nakahara | Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ[END_REF][START_REF] Ribeiro | Insect haemocytes: What type of cell is that?[END_REF][START_REF] Strand | Immunological basis for compatibility in parasitoid host relationships[END_REF]. However, their role in immune processes also depends on the insect species examined [START_REF] Falleiros | Ultrastructural and quantitative studies of hemocytes in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Pyralidae)[END_REF].

The high concentration of PL may possibly be due to their involvement in numerous processes including encapsulation and shaping of the melanotic capsule [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF][START_REF] Nakahara | Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ[END_REF][START_REF] Ribeiro | Insect haemocytes: What type of cell is that?[END_REF][START_REF] Strand | Immunological basis for compatibility in parasitoid host relationships[END_REF], and their production of soluble pattern recognition and antimicrobial proteins [START_REF] Nakahara | Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ[END_REF].

GR and PL act together in defense against macro-pathogens (e.g. parasitoid eggs) during encapsulation [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF][START_REF] Nakahara | Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ[END_REF][START_REF] Ribeiro | Insect haemocytes: What type of cell is that?[END_REF][START_REF] Strand | Immunological basis for compatibility in parasitoid host relationships[END_REF]. Both the concentrations of GR and PL are higher than those of OE and SP, certainly because they can differentiate in OE and SP [START_REF] Beaulaton | Hemocytes and hemocytopoiesis in silkworms[END_REF][START_REF] Han | Hemocytic differentiation in hemopoietic organ of Bombyx mori larvae[END_REF].

GR and PL concentrations were both higher in larvae reared on Gewürztraminer than larvae from others diets and lower in larvae from the control diet than the others diets. The control diet was the most favorable for larvae survival [START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF]. However, larvae reared on Gewürztraminer could have a higher basal level of hemocytes due to the compounds such as phenolic substances found in grape varieties rather than microbes growing in berries [START_REF] Conde | Biochemical changes throughout grape berry development and fruit and wine quality[END_REF][START_REF] Vogelweith | Food-mediated modulation of immunity in a phytophagous insect: an effect of nutrition rather than parasitic contamination[END_REF]. Indeed, in a previous study, we found that controlling microbe abundance in diets did not explain variation in the immune function whereas the presence of berry extracts did [START_REF] Vogelweith | Food-mediated modulation of immunity in a phytophagous insect: an effect of nutrition rather than parasitic contamination[END_REF]. This suggests that variation in immune defenses of E. ambiguella among grape varieties was caused by nutritional differences among host plants rather than microbe abundance.

In agreement with the results of [START_REF] Kim | Up-regulation of circulating hemocyte population in response to bacterial challenge is mediated by octopamine and 5-hydroxytryptamine via Rac1 signal in Spodoptera exigua[END_REF], we found that GR concentration increased after the immune challenge in all diets, thus confirming their important and multiple roles in insect immunity. Additional GR could have two origins. First, GR could be produced by hematopoietic organs and/or derived from other hemocytes already in circulation [START_REF] Nakahara | In vitro studies of hematopoiesis in the silkworm: cell proliferation in and hemocyte discharge from the hematopoietic organ[END_REF][START_REF] Nardi | Embryonic origins of the two main classes of hemocytes -granular cells and plasmatocytes -in Manduca sexta[END_REF]. Second, the immune challenge may induce the mobilization of already existing sessile GR attached to different parts of the insect body [START_REF] Markus | Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster[END_REF][START_REF] King | Spatial and temporal in vivo analysis of circulating and sessile immune cells in mosquitoes: hemocyte mitosis following infection[END_REF]. In our data, we are unable to distinguish between these hypotheses. Only complementary studies such as the one by [START_REF] Markus | Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster[END_REF] on Drosophila melanogaster could explain the origin of these additional hemocytes.

The mechanism by which the concentration of hemocyte is increased upon challenge may have its importance because different mechanisms might be associated with different costs.

Indeed, the costs associated with the production of new hemocytes would likely be higher than the costs of the mobilization of already existing hemocytes.

PL only increased in the control diet and decreased in Gewürztraminer. However, both GR and PL act together in pathogen elimination [START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF]. As mentioned earlier, our control diet is expected to be more nutritive than those made of grapes [START_REF] Vogelweith | Host plant variation plastically impacts different traits of the immune system of a phytophagous insect[END_REF]. Larvae reared on the control diet were probably in much better condition which might have enable them to rapidly produce/recruit more hemocytes in response to the immune challenge. Larvae reared on Gewürztraminer exhibited high basal levels of PL that decreased after the immune challenge to reach that of larvae reared on the control diet. Such depletion might be caused by the intensive use of PL during the immune response that could not be renewed.

OE and SP were the least abundant among the hemocytes (< 5% of the total). OE are responsible for the synthesis and release of the PO enzyme [START_REF] Kanost | Innate immune responses of a lepidopteran insect, Manduca sexta[END_REF], and may be involved in clotting [START_REF] Eleftherianos | Role and Importance of Phenoloxidase in Insect Hemostasis[END_REF][START_REF] Lavine | Insect hemocytes and their role in immunity[END_REF]. The basal OE concentration was not affected by the larval diet, contrary to that of SP. The role of SP is poorly known. [START_REF] Schmitz | The cellular immune response of the Pea Aphid to foreign intrusion and symbiotic challenge[END_REF] have shown that SP are involved in the coagulation process quickly after hemolymph collection. They also suggest that SP participate in energy storage and lipid transport [START_REF] Schmitz | The cellular immune response of the Pea Aphid to foreign intrusion and symbiotic challenge[END_REF]. The effect of diet on SP concentration is the opposite of that on GR concentration. SP are more numerous in larvae reared on Chasselas than in those reared on Chardonnay. In a previous study, we suggested that variation in immune defenses of E. ambiguella among grape varieties is caused by nutritional differences among host plants [START_REF] Vogelweith | Food-mediated modulation of immunity in a phytophagous insect: an effect of nutrition rather than parasitic contamination[END_REF]. Further investigations are needed to determine which compounds of these plant varieties could affect hemocyte types. Following the immune challenge, both OE and SP (such as GR and PL) increased in the control diet. The larvae reared on this diet possess a very low basal level of hemocytes but they are the most able to mobilize or synthesize hemocytes after the immune challenge.

In conclusion, we described four types of hemocyte in E. ambiguella at the 5 th larval instar stage. The concentration of each type of hemocytes was affected by larval diet and an immune challenge. We suggest that larvae can display differential resistance to natural enemies depending on their relative investment in the proportion of each hemocyte type. This result would however need to be confirmed on larvae reared on different grape varieties in natural populations. We predict that larvae having a high proportion of GR will best resist bacteria and parasitoids. Such a finding would be important in the development of future biological control programs focused on the use of parasitoids or entompathogenic microorganisms.

Figure 1 .

 1 Figure 1. Photographs and annotated drawings of the hemocyte types: granulocytes (GR; a,e),

Figure 2 .

 2 Figure 2. Variation among diets in the total concentration of hemocytes prior to and

Figure 3 .

 3 Figure 3. Variation in the concentration of granulocytes (a), plasmatocytes (b), spherulocytes

  Figure 1

  Figure 2

Table 1 .

 1 Diet and challenge effects on the concentration of each hemocyte type (granulocytes, 518 plasmatocytes, spherulocytes and oenocytoids). Significant tests (p-value ≤ 0.05) are shown in 519

	520	bold.
		Hemocyte type		χ²	Df	p-value
		Granulocytes	Diet	4.45	3;	0.006
			Challenge	29.90	1;	< 0.0001
			Diet * Challenge	2.17	3;	0.10
		Plasmatocytes	Diet	1.46	3;	0.23
			Challenge	0.19	1;	0.66
			Diet * Challenge	3.59	3;	0.02
		Spherulocytes	Diet	19.66	3;	< 0.0001
			Challenge	1.48	1;	0.23
			Diet * Challenge	3.72	3;	0.01
		Oenocytoids	Diet	1.81	3;	0.15
			Challenge	0.73	1;	0.40
			Diet * Challenge	2.92	3;	0.04

Acknowledgments

We thank Lionel Delbac and Bastien Quaglietti for their technical assistance, Juliette Poidatz for drawings, and Maximilian Körner and Jos Kramer for proof-readings. This study was supported by the CNRS and grants from the ANR (ANR-07-JCJC-0134 and ANR-08-JCJC-0006). The fourth author's research is part of the Labex Cote research programme.

The authors declare no competing financial interests.