F Fines-Guyon

F Guerin

F Ethuin

J Borgey

S Lubrano

Le Hello
email: lehello-s@chu-caen.fr

Hello S Le

Prof Le Hello Simon

Investigation of Serratia marcescens surgical site infection outbreak associated with peroperative ultrasonography probe

Keywords: Surgical site infections, Serratia marcescens, Whole-genome sequencing

published or not. The documents may come

Introduction

Serratia marcescens is an opportunistic pathogen that can cause a wide variety of healthcare-associated infections, such as respiratory and urinary tract infections, meningitis, bacteraemia and surgical site infections (SSIs), particularly in settings such as intensive care units (ICUs) [START_REF] Mahlen | Serratia infections: from military experiments to current practice[END_REF][START_REF] Cristina | Serratia marcescens infections in Neonatal Intensive Care Units (NICUs)[END_REF] . This Gram-negative bacteria can easily spread in the environment and contaminate medical equipment [START_REF] Mahlen | Serratia infections: from military experiments to current practice[END_REF][START_REF] Cristina | Serratia marcescens infections in Neonatal Intensive Care Units (NICUs)[END_REF] , drugs [START_REF] Mahlen | Serratia infections: from military experiments to current practice[END_REF][START_REF] Cilli | Serratia marcescens sepsis outbreak caused by contaminated propofol[END_REF] , antiseptic solutions [START_REF] Merino | Serratia marcescens bacteraemia outbreak in haemodialysis patients with tunnelled catheters due to colonisation of antiseptic solution. Experience at 4 hospitals[END_REF] , or the hands of caregivers [START_REF] Mahlen | Serratia infections: from military experiments to current practice[END_REF] .

Investigations usually find, as a surgical environment reservoir, razors [START_REF] Leng | Outbreak of Serratia marcescens postoperative infection traced to barbers and razors[END_REF][START_REF] Kim | Outbreak investigation of Serratia marcescens neurosurgical site infections associated with a contaminated shaving razors[END_REF] , detergent-disinfectant solutions [START_REF] Ehrenkranz | Antibiotic-sensitive Serratia marcescens infections complicating cardiopulmonary operations: contaminated disinfectant as a reservoir[END_REF] or saline solutes [START_REF] Pegues | Serratia marcescens surgical wound infection following breast reconstruction[END_REF] . In August 2018, four SSIs due to S. marcescens occurred in a digestive surgery ward in a French teaching hospital. This led to extended investigations in order to identify the origin of the outbreak.

Methods

This study took place in the teaching hospital of Caen, a 1,410-bed hospital located in Normandy, France. On 6 August 2018, a fourth spatiotemporal case of S. marcescens SSI appeared in the digestive surgery ward. Furthermore, the clinical strains displayed the same antibiotic resistance profile. As a consequence, the hygiene and infection control department of the hospital was alerted on 7 August 2018 by surgeons, microbiologists and intensive care seniors; this led to the immediate closure of the operating room. To screen unnoticed cases since the beginning of 2018, retrospective investigations were performed. Information was collected from the acts traceability booklet and medical files, as well as coding data transmitted by the hospital archive department, the microbiology laboratory and the hospital pharmacy. Cases were defined as follows: any patient who had undergone digestive surgery in the incriminated operating room, especially a hepatic tumour resection by radiofrequency or by hepatectomy (with intraoperative ultrasound guidance) and who had a microbiological sample positive for S. marcescens. The National Nosocomial Infections Surveillance (NNIS) risk index was calculated for cases. [START_REF] Emori | National nosocomial infections surveillance system (NNIS): description of surveillance methods[END_REF] Environmental samples were performed in the operating room, including airlock by taking air samples (impact biocollector) and surface samples (contact agar plates), in order to find the reservoir of S. marcescens. Isolated strains were identified using the MALDI-TOF mass spectrometry method (Bruker ® , Germany). Antimicrobial susceptibility was tested against a large panel of antibiotics according to French and European guidelines 10 for all S. marcescens strains, clinical and environmental, isolated throughout this investigation, as well as two supplementary not-linked strains. Moreover, these strains were subjected to whole-genome sequencing (WGS) using a NextSeq 500 platform (Illumina, USA), generating 150 bp paired-end reads. Genomes were de novo assembled using SPAdes v 3.12 software. Every genome is available on the NCBI BioProject: PRJNA648275. All known acquired antibiotic resistance genes were screened thanks to the Resfinder database (www.cge.dk). Furthermore, point mutations were searched in order to detect alterations in the quinolone resistance-determining regions (QRDRs). Reads were mapped against the reference genome S. marcescens subsp. marcescens Db11 (Nucleotide accession number: NZ_HG326223) using the Burrows-Wheeler Aligner, and single nucleotide polymorphisms (SNPs) were determined in order to evaluate the distance between all the sequenced strains using mpileup, as previously described. 10

Results

A total of eight patients suffered from SSIs caused by S. marcescens between 5 April and 7 August 2018 in the same digestive surgery ward. These patients underwent hepatectomy with intraoperative ultrasound guidance, with four of them undergoing tumour resection by radiofrequency. All eight cases had an early deep SSI (between 0 and 11 days after surgery), and all but one case received adapted antibiotic treatment for 11 to 42 days. Three cases required surgical revision to control the infection (see patients characteristics in Table 1). In all cases, the drug used for preoperative surgical antibiotic prophylaxis and method of administration were in accordance with in situ and French Society of Anaesthesia and Intensive Care Medicine guidelines, which consists of 2 g cefazolin injection [https://sfar.org/]. The NNIS score was variable among patients: 0 (n = 3), 1 (n = 4) and 2 (n = 1). The highest score corresponded to the index case who suffered from deterioration in his general status and potentially contaminated surgery. His biliary tract infection (caused by S. marcescens) was responsible for immediate postoperative septic shock, which was treated with a combination of cefepime and gentamicin for 25 days.

The investigation of the eight cases revealed two common points: i) the operating room and ii) the use of a unique T-shaped intraoperative probe 8816 (BK Ultrasound, Peabody, Massachusetts). The operating room was closed, and radiofrequency material was retired on 7

August 2018, whereas the ultrasound probe was sequestrated three days after the alert. In fact, time was needed to analyse 67 environmental samples, including surgical material samples (radiofrequency material and T-shaped probes), medicine fridges, scopes, room surfaces (floors, walls, materials, trolleys, operation tables, cables, scyalitic lamps, and cans), detergentdisinfectant and antiseptic solutions, syringe drivers, ultrasound scanners, and computer keyboards. Surgical interventions were performed by several surgical teams; hence, no S. marcescens carriage testing was performed on caregivers. First, none of the 67 samples were positive for S. marcescens. A new inspection was performed on the ultrasound probe by the hygiene microbiologist. It turned out that the sleeve that allowed for the seal between the cable and body of the probe was unstuck, facilitating the probable formation of a reservoir. In addition, an analysis of the local practices highlighted two major non-compliances. First, the probe was used intraoperatively without a single-use sterile protective sheath, and second, the maintenance carried out after its use was not in conformity (absence of terminal disinfection after cleaning).

Thanks to these observations, new samples were taken by modifying the sampling technique in pre-adding a step of sterile water immersion/injection of/in the probe, in particular in the sleeve. Doing so, a S. marcescens strain was isolated and displayed the same pattern of antibiotic resistance as those isolated from the eight patients. All these epidemic strains harboured the chromosomal AmpC bla-SRT-2 and aac(6')-Ic genes, conferring resistance to most penicillins, including cefazolin and amikacin, respectively. Epidemic strains also carried the bla-TEM gene, coding a penicillinase; aminoglycoside aadA1 gene, conferring resistance to streptomycin and spectinomycin; and dfrA1 gene, leading to resistance to trimethoprim and co-trimoxazole.

QRDRs were chromosomally substituted in GyrA (Ser83Ile and Asp87Asn) and ParC (Ser80Ile), conferring high-level resistance to fluoroquinolones through three amino acid changes. Epidemic strains remained susceptible to key antibiotics for S. marcescens treatment (i.e., third-generation cephalosporins or carbapenem). In silico analyses confirmed that all eight clinical strains were closely related to the probe strain, with an average of distance of 9 SNPs (min: 1, max: 24) inside the cluster, while a distance 87 000 SNPs split the epidemic strains with two not-linked strains (see Supplemental Figure ).

In total, the incriminated T-shape probe was used in nine interventions between the index case and its withdrawal. Among them, eight patients presented with SSIs caused by S. marcescens. In the only patient who did not contract an SSI, the contaminated probe was used with a very short contact time. Regarding surgical procedures, numerous non-compliances were uncovered, such as the absence of biocleaning and upkeep traceability for radiofrequency material, T-shape probes and fridges, as well as the absence of the single-use sheath of the probe during ultrasonography.

Not one case of S. marcescens SSI has been reported since the end of this investigation and corrective measures have been taken, such as medical device vigilance, staff formation and procedure adjustments in biocleaning and traceability and the use of the probe sheath during deep surgical operations.

Discussion

This S. marcescens outbreak investigation revealed eight postoperative infections due to the use of a contaminated T-shaped intraoperative probe. During ultrasonography, the probe was used in direct and long contact with tissues without a single-use sheath. These defaults could explain a brief five day-mean elapsed time between the operation and the first signs of infection. This delay is shorter than that described in other publications. [START_REF] Fernández | Clinical study of an outbreak of postoperative mediastinitis caused by Serratia marcescens in adult cardiac surgery[END_REF][START_REF] Ulu-Kilic | Outbreak of postoperative empyema caused by Serratia marcescens in a thoracic surgery unit[END_REF] Interestingly, our investigation disentangled this epidemic episode by detecting all patients (including the index case), by identifying the cause of the SSI and the mechanism related to the formation of the reservoir. No similar example was found in the literature. Indeed, in most of studies referring to S. marcescens SSI outbreaks, a strain reservoir was epidemiologically suspected, rarely confirmed, or the number of contaminated patients were estimated. [START_REF] Mahlen | Serratia infections: from military experiments to current practice[END_REF][START_REF] Cristina | Serratia marcescens infections in Neonatal Intensive Care Units (NICUs)[END_REF][START_REF] Cilli | Serratia marcescens sepsis outbreak caused by contaminated propofol[END_REF][START_REF] Merino | Serratia marcescens bacteraemia outbreak in haemodialysis patients with tunnelled catheters due to colonisation of antiseptic solution. Experience at 4 hospitals[END_REF][START_REF] Leng | Outbreak of Serratia marcescens postoperative infection traced to barbers and razors[END_REF][START_REF] Kim | Outbreak investigation of Serratia marcescens neurosurgical site infections associated with a contaminated shaving razors[END_REF][START_REF] Ehrenkranz | Antibiotic-sensitive Serratia marcescens infections complicating cardiopulmonary operations: contaminated disinfectant as a reservoir[END_REF][START_REF] Pegues | Serratia marcescens surgical wound infection following breast reconstruction[END_REF][START_REF] Emori | National nosocomial infections surveillance system (NNIS): description of surveillance methods[END_REF][START_REF] Gravey | RamR deletion in Enterobacter cloacae complex as a consequence of therapeutic failure during a long-term hospitalized patient[END_REF][START_REF] Fernández | Clinical study of an outbreak of postoperative mediastinitis caused by Serratia marcescens in adult cardiac surgery[END_REF][START_REF] Ulu-Kilic | Outbreak of postoperative empyema caused by Serratia marcescens in a thoracic surgery unit[END_REF] This investigation also highlighted that the sole use of contact agar plates was not sufficient to find S. marcescens on the probe. If a specific device is suspected to be infected, we recommend the use of the technique presented in this report, namely immersion of the device in sterile water and/or injection of sterile water into unreachable nooks. Furthermore, WGS is a highly discriminative tool widely used in large scale epidemiologic investigations [START_REF] Kim | Outbreak investigation of Serratia marcescens neurosurgical site infections associated with a contaminated shaving razors[END_REF][START_REF] Gravey | RamR deletion in Enterobacter cloacae complex as a consequence of therapeutic failure during a long-term hospitalized patient[END_REF] , and we showed through our work that it is valuable to ascertain our hypothesis of contamination.

Our investigation raised several questions concerning care practices [START_REF] Carrico | Ultrasound probe use and reprocessing: Results from a national survey among U.S. infection preventionists[END_REF] . It must be emphasized that high surgical activity leaves little time for the caregivers: i) to allow a good check-up of the equipment and a realization of an adapted upkeep and ii) to get practical training courses and to master the procedure. In addition, the use of the ultrasound probe without a sheath is a common practice by surgeons to obtain a better surgical view, but it is not without risks in terms of health-associated infections. Patients contamination by the T-shaped probe could depend on both bacteria inoculum and duration of exposure and could not be avoided by the recommended antibiotic prophylaxis (cefazolin 2 g), as it is ineffective on S. marcescens.

We would like to underline that these results were obtained through a close cooperation between the hospital hygiene department, the digestive surgery ward, the hospital archive department, and the microbiology laboratory. This led to the issuing of a medical device alert and to the withdrawal of the T-shaped probe and showed that the use of a single-use sterile sheath placed on the tip of probe during ultrasonography in laparoscopic liver resection is essential in order to avoid such peroperative infections. Infection prevention and control measures, including reminder courses on adequate cleaning and decontamination of the probe were set up. Since then, no other case was observed.

Conclusion

We reported an outbreak of S. marcescens SSIs associated with peroperative ultrasonography. These results led to the withdrawal of the probe, to a reinforcement of the training course on the pre-cleaning step, in particular disinfection procedures vigilance, and to a

correction of the practices, especially by the systematic use of a single-use cover during peroperative ultrasonography in laparoscopic liver resection.

Acknowledgements

We thank Annie Lemarié, Nelly Devêre, Valerie Moreno and Florence Souplet for their excellent technical assistance work, and the administration and infection control staff, as well as the pharmacy hospital, for their support during the investigation.

Funding

No support

Conflict of interest

All authors: No reported conflicts. Table 1. Operating history and patient characteristics of Serratia marcescens surgical site infections, 2018, teaching hospital of Caen, Normandy, France.

Tables


Katia Chirkova Folk

teaching and research institutions in France or abroad, or from public or private research centers.

• ⾼度濒危,⻓期处于语⾔接触与语⾔兼⽤状态 • 多续话的使⽤⼈群出现了忘词和语⾔变异等突出现象, 对记录研究多续话造成困难 • 在诸多川⻄南语⾔研究中,多续话得到较少关注,且少 有相关的公布语料,尤其缺乏对多续话退出⽇常交际之 前的记载 • 在没有早期语料能够参考的情况下,很难评估从以上多 续话的"不完全语⾔使⽤者"(semi-speaker)那⾥得到的 语料的真确性和代表性,也⽆法完整了解多续话的语⾔ ⾯貌 总结 吴万才 《冕宁县藏族多续之谱》 • ⾃从1955年开始编写 • 历经⼏次修改,早期版本名为《吴哩⽒族谱》 (1980年) • 因为居住在越⻄、⽢洛的扎拉洼与冕宁的吴⽒族⼈ 认为他们同属于⼀个"潘策",所以决定合写族谱 • 通过这个家谱的建构,⽢洛、越⻄"尔苏⻄番"扎拉洼 家族与冕宁等地的"多续⻄番"吴哩家族联系到了⼀起 《冕宁县藏族多续之谱》 • 吴万才于1926年出身于冕宁⼤桥埃基村 • 由于吴万才⻅到藏族多续⼈的⺠族语⾔、⺠⻛⺠ 俗、⺠族传统⽂化逐渐被遗忘,故其从1955年开始 收集记录多续⽂化并将材料编辑为书 • 吴万才表示他著书的意义在于: • 上则续本⺠族的美好传统⽂化、语⾔、⼜继承了祖 先们的⻛俗习惯等,下则传后代,不忘根本,不变 其质,保留着本⺠族⼀点特⾊,但愿我本⺠族⼈⼈ 醒之,共同提倡 吴万才⼿稿 • 全书248⻚45500个字;⼿稿⽤汉字记⾳的⽅式记录下了 多续话词汇与⻓篇语料 • ⼿稿包含2700多词条的多续话词汇表和⼀些传统歌词、 宗教仪式念词、谜语、成语、传统故事等语料,是⽬前 为⽌词汇量与范围最⼴的多续话语料集 • 因为该⼿稿从1955年起开始编写,故其主要反映了20世 纪中下叶的多续话使⽤情况,这为研究较早期的多续话 提供了可能 词汇范围 • 庙宇、神类 • ⼈类、残疾⼈类 • ⼈体类、称呼类 • 服饰类 • 树⽊类、草类、⽵类、农作物类 • 房屋类 • ⼯匠类 • 颜⾊、官品、病、药四类 • 官品类,疾病类、药材类 • ⽤具类、数⽬类 • ⽅位名、睡⼆类,地名类 • 星⾠、⽇⽉、⽔三类 • ⽔、⽕、江、河、桥、海,六类 • ⼭、⽯⼆类,时令类 • ⻛、云、雷、⾬,四类 • 纸、笔、墨、砚、书,五类 • 粮⻝、蔬菜、味道,三类 • ⾍、禽,⼆类;兽、油,⼆类;⾦、 银、铜、铁、锡、宝、钱,七类 词汇范围 • 杂句类 • 成句词类 • 乐器、歌舞、骂⼈三类 • 名⼈录,戳(⼝字旁,⽔字旁)爹嘎 不的故事 • 官话、九⻰哩汝话,吹⽕烫神寺 • 拉摸⾐话 • 藏族的故事类 • 杂句类 • 冕宁县⼟千百户 ⺟,迷吸嵌拖,【⼝+铁】咪杜㕷,哦以哦,㕲虾以 㕲虾。【⼝+按】以五⾜⼝,【⼝+杀】蚂举【⼝+ 料】【⼝+料】写,【⼝+刘】⻁以【⼝+铁】,【⼝ +塔】谢,【⼝+料】【⼝+料】⽐拿。阿迷嘞,【⼝ +按】𠮨以五⾜【⼝+杀】蚂,举【⼝+料】【⼝+料】 写。猓兹咔【⼝+擦】嘞,⾜迷【⼝+刘】嘞,⼥破 【⼝+摆】拿拿。㕷【⼝+切】迷嘎,舍迷吸拿,萨 姑⽶,以㕷【⼝+切】不,吴【⼝+万】嘞,拿愚嘎、 ⼋嘎、⼥⻣,也哠嘞吸。萨姑咪以雅喀㕷拿嘞,愚 嘎、⼋嘎,阿妈拿拿,贾不迷取,愚嘎,贾不取, ⼋嘎嵌拖,【⼝+塔】咔咔,【⼝+按】以阿妈麻⼰。 【⼝+濯】麻苏。𠮨【⼝+按】以阿麻⼰,【⼝+按】 以阿妈,库哠,库⽉㕷,【⼝+濯】【⼝+绕】。【⼝ 含义 注⾳汉字 多续今⾳ 吴万才⼿稿 国际⾳标记⾳ • 经过在多续地区进⾏的三年调查,我们搜集了吴万才词汇表上70%多的词 汇,这对系统研究其⼿稿中的多续语料奠定了基础 • 通过研究⽐较发现,吴万才的汉字记⾳有系统性、⼀致性与准确性的特征 注⾳汉字 551 241 汉字 ⾃创字 研究⽅法 • 建⽴注⾳汉字与多续今⾳的对照库 • 统计吴万才⼿稿中注⾳⽤的汉字和⾃创字,并分析每个注 ⾳汉字与其对应的多续今⾳之间的关系,进⽽归纳出⼿稿 汉字标⾳的规律 • 将归纳出的规律⽤到分析没有对应的多续今⾳的注⾳汉字 与⾃创字,恢复部分已丢失的词汇 • 基于归纳的特征初步探讨吴万才⼿稿所反映的多续语⾳与 现代多续话语⾳的差别 基础⽅⾔ • 吴万才注⾳时以他的⺟语⻄南官话冕宁县汉语⽅⾔(冕宁 话)读⾳为基础,这可以通过其注⾳词汇选择⽽看出 • ⼿稿使⽤了⼀系列较典型的⻄南官话特征词 • '尿泡'='膀胱'、'腰⼦'='肾'、'寡公⼦'='鳏夫' • 四川⽅⾔字注⾳: • 𤆵 pʰa⁴⁴ '软'、 naŋ⁴⁴ '瘦⼩或细⻓'、 kʰa²¹/tɕʰa²¹ '跨' • 冕宁话中的特殊⾳变: • 北京的tʂ-,tʂʰ-,ʂu,ʐu-声⺟的u韵字,分别读为pf, pfʰ,fu,vu,f-,如猪:pfū,桌: pfō,说:fó 等 基础⽅⾔ • 《冕宁县志》(2009: 747-748) 冕宁话调查 • 为了能够准确地判断⼿稿注⾳汉字的冕宁读⾳与多续话今 ⾳的对应情况,我们采录了吴万才⼿稿⽤于记录多续话的 551个普通汉字与241⾃创字的⾳符字 • 因为冕宁话尚未被系统研究过,且现代发⾳与《冕宁县 志》中的读⾳略不同,因此⽤严式国际⾳标,⼀律按照实 际发⾳标⾳ 注⾳系统介绍 • 通过⻓时间紧密接触,多续话有向冕宁话⾳靠拢的趋势, 声⺟发⾳部位、⽅法基本相同 冕宁话与多续话:4个声调 冕宁话韵⺟⽐多续话多 冕宁话声⺟⽐多续话少 ⾳节结构 冕宁话 (C)(j/w)V(n/ŋ)T 多续话 (N)(C)(j/w)V T 声调对应举例 声⺟对应关系 ⾃创字 • 对应于多续话有⽽冕宁话所没有相对应的声⺟、韵⺟、声调 的组合(⾳节),吴万才⼿稿就采⽤形声⼿段创造新汉字, 借⽤与所标⾳的多续⾳节⾳同或者⾳近的⾳节(普通汉字) 当作表⾳的⾳符 • 这些字都是由⼀个标⾳的⾳符和⼀个不标⾳的义符组成的 ⾃创字 1. ⼝字旁,占⾃创字94%,标⾳不表意,如【⼝+歪】, 【⼝+⽩】,对应于与⾳符声调、声⺟或韵⺟不同的多 续话⾳节 2. 其他义符,共12个,如【亻+当】,【扌+慈】等。基 于有多续今⾳对应的例⼦,可以假设这⼀类⾃创字⾳符 的读⾳与多续今⾳相同,⽽义符⽤于表示类属
• 对该⼿稿的整理研究不仅为我们提供了上世纪中下叶多 续话较为全⾯的⾯貌,也弥补了早期多续语料不⾜的缺 陷

• 汉语是影响⻄南少数⺠族语⾔的主要因素之⼀

• ⺠族⾛廊中,少数⺠族语⾔研究离不开当地汉语⽅⾔, ⺠间⽂献,研究⽅⾔也离不开少数⺠族语⾔

  of the Tosu-Chinese Vocabulary, Tosu I-Yu: The Structure and Lineage of Tosu, A New Language》. 京都: 松⾹堂.

  and Katia Chirkova. 2016. « Duoxu tonal developments in Tibeto-Burman context ». STLS-2016, Seattle • ⻩布凡&尹薇彬 (2012: 60): ae-a • 注⾳汉字:a -an • 肝--嘎;嵌--咔;丹--搭… • 冕宁话: -an [aen], 肝 [kaen⁴⁴]• '樟脑', WT ག་,ར་,"肝都" kaen⁴⁴tu⁴⁴ • '说': kʰa³³tʰo³³, "嵌拖" kʰaen⁴⁴tʰo⁴⁴ > 拟测⾳ kʰae³³tʰo³³• '⼝': kʰa³³pi³³, "嵌吡" kʰaen⁴⁴pi⁴⁴ > 拟测⾳ kʰae³³pi³³ 了解多续话语⾳演变情况


Lénaïc B Cadet
email: lenaic.cadet@univ-lyon2.fr

Hanna Chainay
email: hanna.chainay@univ-lyon2.fr

How preadolescents and adults remember and experience virtual reality: the role of avatar incarnation, emotion, and sense of presence

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

Introduction

Embodied cognition has given the body a central role in cognitive processes (e.g., memory, attention, emotion, action). More specifically, Barsalou [START_REF] Barsalou | Grounded Cognition[END_REF] proposed that the state of the body, the situation of the action, and the modality of the simulation govern cognition.

Evidence of embodied cognition has been demonstrated in children too, with empirical studies demonstrating that cognition emerges from sensorimotor interactions of the child with the environment [START_REF] Kiefer | Embodiment theory and education: The foundations of cognition in perception and action[END_REF][START_REF] Ruiz | Motor Skills, Motor Competence and Children: Bruner's Ideas in the Era of Embodiment Cognition and Action[END_REF][START_REF] Wellsby | Developing embodied cognition: insights from children's concepts and language processing[END_REF].

In a virtual reality (VR) context, it is possible to create a fully virtual sensorimotor environment that the user can explore, and in which he or she has the possibility of embodying an avatar (i.e., a virtual body). It is critical for research on the child and adolescent population to understand the effect of embodying an avatar due to their early exposure to multiple virtual representations of themselves in different medias (e.g., picture on mobile phone, digital camera, handheld game console [START_REF] Bailey | Chapter 9 -Immersive Virtual Reality and the Developing Child[END_REF]), despite the ongoing development of the self. VR is a specific media that allows users to embody virtual avatars and to evolve in a virtual environment in the firstperson view, especially with a head-mounted display (HMD), allowing visuomotor synchronicity. Research on adults has shown that VR is indeed an effective device to create feelings of embodiment [START_REF] Kilteni | The Sense of Embodiment in Virtual Reality[END_REF] or to explore the embodied nature of memory [START_REF] Repetto | Virtual Reality as an Embodied Tool to Enhance Episodic Memory in Elderly[END_REF]. However, only a few studies have investigated this point in preadolescents (i.e. middle school pupils, e.g., [START_REF] Mikropoulos | Presence: a unique characteristic in educational virtual environments[END_REF][START_REF] Mikropoulos | Factors That Influence Presence in Educational Virtual Environments[END_REF]). Thus, in order to ensure respectful use of VR in this population whose access to this media will potentially grow in the comings years, more studies are necessary to explore the effects of embodying an avatar in VR on cognition. The purpose of the present study is to compare adult and preadolescents, middle school pupils (MSP) with regard to the effects of embodying a virtual avatar during a VR experience on different aspects of cognition assumed to be linked to self-representation: memory, emotion, and sense of presence.

Avatar: effects on memory and presence

Presence is defined as the perception of being there in a physical or virtual environment [START_REF] Slater | A note on presence terminology[END_REF]. On one hand, research on adults has revealed that the incarnation of a virtual body (i.e., an avatar) can enhance presence [START_REF] Slater | Simulating virtual environments within virtual environments as the basis for a psychophysics of presence[END_REF], and memory [START_REF] Repetto | Virtual Reality as an Embodied Tool to Enhance Episodic Memory in Elderly[END_REF]. On the other hand, some studies on preadolescents MSP have shown the same positive impact of avatar incarnation on presence [START_REF] Mikropoulos | Presence: a unique characteristic in educational virtual environments[END_REF][START_REF] Mikropoulos | Factors That Influence Presence in Educational Virtual Environments[END_REF]. Some studies have investigated this in adults (e.g., [START_REF] Steed | The impact of a self-avatar on cognitive load in immersive virtual reality[END_REF][START_REF] González-Franco | The contribution of realtime mirror reflections of motor actions on virtual body ownership in an immersive virtual environment[END_REF][START_REF] Pan | How Foot Tracking Matters: The Impact of an Animated Self-Avatar on Interaction, Embodiment and Presence in Shared Virtual Environments[END_REF]), however concerning younger populations research on avatar incarnation has predominantly focused on children or adolescents with neurodevelopment disorders (e.g., autism disorder [START_REF] Bellani | Virtual reality in autism: State of the art[END_REF]).

First, concerning the relationship between presence and embodiment in an avatar, for MSP, development could have a critical role. In fact, research on adults has, at least partially, attributed the state of presence to the virtual implication of the self [START_REF] Ratan | Self-Presence, Explicated: Body, Emotion, and Identity Extension into the Virtual Self, Handb[END_REF]. Bailey and Bailenson [START_REF] Bailey | Chapter 9 -Immersive Virtual Reality and the Developing Child[END_REF] discussed the development of the self by pointing out that, even if a child is able to recognize themselves in a mirror (mirror task) at the age of 3-4 years, it does not mean that the self is fully mature. In fact, Suddendorf and Butler [START_REF] Suddendorf | The nature of visual self-recognition[END_REF] argued that children who succeeded in the mirror task were not always able to recognize themselves in a video. With older children (6-7 years), Segovia and Bailenson [START_REF] Segovia | Virtually True: Children's Acquisition of False Memories in Virtual Reality[END_REF] found that when children saw a virtual version of themselves swimming with orcas, they confused it with reality, and later recalled that it had happened in real life. However, Calvert et al. [START_REF] Calvert | Preadolescent girls' and boys' virtual MUD play[END_REF] have shown that preadolescent's behavior (mean age of 10 years and 11 month) was not affected by incarnating an avatar having the opposite sex that their own sex. Thus, the self is a complex mechanism allowing us to recognize ourselves in the present, but also at different points of time and space. Attention was drawn on the fact that VR is a peculiar context that could be challenging for the self, even for adults [START_REF] Bailey | Chapter 9 -Immersive Virtual Reality and the Developing Child[END_REF]. In VR contexts, the virtual self can diverge from the user's self in terms of looks and attitudes. Thus, because of the different levels of maturation of the self, embodying an avatar in VR could have different impacts on emotion or on sense of presence in MSP than in adults.

Second, concerning the link between embodiment and memory, studies have shown positive effects of virtual embodiment on episodic memory performance in young adults (see for review, [START_REF] Tuena | How virtual embodiment affects episodic memory functioning: A proof-of-concept study[END_REF]). A possible explanation can be found in neurodevelopmental observations that functional episodic memory is linked to the development of locomotion, and the alignment of hippocampal place cells and grid cells with the environment [START_REF] Glenberg | Contribution of Embodiment to Solving the Riddle of Infantile Amnesia[END_REF]. Moreover, the enactment effect has already demonstrated that a possible action of the body on an object, even imaginary, can impact the memory of this object. As an example, Dutriaux and Gyselinck [START_REF] Dutriaux | Learning Is Better with the Hands Free: The Role of Posture in the Memory of Manipulable Objects[END_REF] showed that better memory performances for a list of manipulatable objects were obtained when the participants' hands were free during encoding, compared to when they had to keep their hands crossed behind their back. In a VR context, it is possible that embodying an avatar in the firstperson view (with full-body functioning), compared to seeing nothing, enhances possible actions on the virtual environment, and thus enhances the memory thereof. Ratan [START_REF] Ratan | Self-Presence, Explicated: Body, Emotion, and Identity Extension into the Virtual Self, Handb[END_REF] mentioned that the effects of embodying a virtual body on cognitive functions could depend on the level of self-presence, in other words, how much the user feels connected to their virtual self-representation on different levels (body, emotion, identity). Moreover, it has been pointed out for many years that self-reference could positively influence encoding and retrieval in memory processes [START_REF] Rogers | Self-reference and the encoding of personal information[END_REF]. So, it is possible that this effect is stronger in adults than in MSP, in view of the still-maturing body schema and self in the latter population. In order to explore such a possibility, in this study, we manipulated the embodiment by exposing participants to virtual environments through an avatar seen in the first-person view, or through a non-body condition, where the participant could only see the two VR controllers.

Emotional nature of the virtual environment: effects on memory and presence

Research on adults found that presence can be impacted by emotion [START_REF] Baños | Immersion and Emotion: Their Impact on the Sense of Presence[END_REF][START_REF] Gromer | Causal Interactive Links Between Presence and Fear in Virtual Reality Height Exposure[END_REF][START_REF] Riva | Affective Interactions Using Virtual Reality: The Link between Presence and Emotions[END_REF]. Some empirical findings support that emotion can enhance presence in child populations too [START_REF] Baumgartner | Neural correlate of spatial presence in an arousing and noninteractive virtual reality: an EEG and psychophysiology study[END_REF][START_REF] Baumgartner | Feeling present in arousing virtual reality worlds: prefrontal brain regions differentially orchestrate presence experience in adults and children[END_REF].

However, because emotional responses and regulation are still developing during childhood and adolescence [START_REF] Michalska | The psychobiology of emotional development: The case for examining sociocultural processes[END_REF], it is possible that the strength of the link and the relationship between emotion and presence is not the same for preadolescents and adults. In order to explore these relationships, we manipulated emotion systematically by presenting positive, negative, and neutral stimuli in the virtual environment. In order to measure emotional responses precisely, we used self-assessed questionnaires (Self-assessment Manikin, [START_REF] Bradley | Measuring emotion: the Self-Assessment Manikin and the Semantic Differential[END_REF]) administered immediately after exposure to the virtual environment, which we coupled with physiological measures during exposure, as previous studies did in their VR protocols [START_REF] Gromer | Causal Interactive Links Between Presence and Fear in Virtual Reality Height Exposure[END_REF][START_REF] Felnhofer | Is virtual reality emotionally arousing? Investigating five emotion inducing virtual park scenarios[END_REF].

The enhancing impact of emotion on memory is a well-known phenomenon in the literature on adult [START_REF] Kensinger | Memory and emotion[END_REF] and child populations [START_REF] Michalska | The psychobiology of emotional development: The case for examining sociocultural processes[END_REF][START_REF] Bradley | Measuring emotion: the Self-Assessment Manikin and the Semantic Differential[END_REF][START_REF] Felnhofer | Is virtual reality emotionally arousing? Investigating five emotion inducing virtual park scenarios[END_REF], but in the context of virtual reality, more studies are necessary to describe the respective effects of emotion and presence on memory performances. Makowski et al. suggested that the impact of emotion on memory depends on presence [START_REF] Makowski | Being there" and remembering it: Presence improves memory encoding[END_REF]. In their study, with young adults population, they measured emotion and memory in relation to scenes from a movie. Thus, the emotion was not methodically manipulated, and did not depend exclusively on the nature of the stimulus recalled later. To our knowledge there is no study that investigated effect of emotion on memory in VR with preadolescents. Thus, in our study, we asked participants to recall as many stimuli as possible, and the emotional stimulation during our protocol mostly stemmed from the stimuli themselves. In addition, the narrative point of view during the film (third-person view) did not allow participants to develop presence in the same way that a first-person view could. Riva et al. [START_REF] Riva | From intention to action: The role of presence[END_REF] described three levels of presence: proto presence, or the capacity to set perception and movements together and discern the internal and the external environment; core presence, the capacity to focus sensorial processes on the current task; extended presence, the capacity to select previous experiences as relevant for the self. In view of this proposition, a first-person view, especially with a full-body avatar, provides the best conditions for enhancing presence.

In our study, we should be able to explore the combined effects of the emotional nature of the virtual environment and the embodiment of an avatar, not only on memory, but on sense of presence, and emotional subjective and physiological responses.

Aim of the study

To summarize, the first goal of this study is to explore the respective roles of three factors (i.e., avatar embodiment, emotional responses, and sense of presence) on memory performances in preadolescents, and to compare these results to adults' ones. To do so, we used two conditions of embodiment: a full-body avatar in the first-person view versus only the view of the two controllers at their actual location (i.e., avatar versus no-avatar). We hypothesized that emotional enhancement of memory (EEM) would be stronger in preadolescents than in adults, and that embodying an avatar would enhance this effect, especially in preadolescents.

The secondary objective of this study is to investigate the impact of avatar embodiment, age, and the emotional nature of the virtual experience on sense of presence and emotional responses (subjective, behavioral, and physiological). To achieve this secondary objective, we used different measures provided by the HMD (head position and rotation), and additional material (collecting heart rate and pupillary dilation during the task). We predicted that visiting a place with emotional stimuli through an avatar would elicit a more heightened sense of presence, emotional responses, and memory performances than visiting it with no avatar. We also expected these same effects of avatar for the places with neutral stimuli, but these effects would be decreased compared to the environments with emotional stimuli.

Method

Participants

Seventy-two participants divided into two age-groups took part in the experiment: thirty-two preadolescent MSP (Mage = 12.1 years, range = 10-14 years; 19 girls and 13 boys) and forty young adults, students at Lyon 2 University (Mage = 20.39 years, range = 18-28 years; 20 women and 20 men). There were four experimental groups: preadolescents with avatars in the virtual environment (N=16), preadolescents with no avatar (N=16), young adults with avatars (N=20), and young adults with no avatar (N=20). All preadolescents were pupils attending middle school in Lyon and Clohars-Carnoët. All participants (and/or their legal representatives) gave their written informed consent in accordance with the Helsinki declaration, and had declared having good hearing and vision. The research was approved by Ethics Committee (Comité de Protection des Personnes, Ile de France, Nr. 2018 A01669-46).

Our target sample size was determined using an a-priori power analysis (G*Power [START_REF] Faul | G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences[END_REF]), using η²p from previous studies investigating memory and sense of presence for virtual reality environments. For example, previous studies [START_REF] Cadet | Memory of virtual experiences: Role of immersion, emotion and sense of presence[END_REF][START_REF] Buttussi | Effects of Different Types of Virtual Reality Display on Presence and Learning in a Safety Training Scenario[END_REF] found that η²p ranged from .038 to .219 for memory and from .038 to .538 for sense of presence. Using a η²p =.16, our study design with two between-participants factors (age and avatar condition) and one repeated factor (valence), could achieve 80% power with 16 young adults and 16 preadolescent participants per group.

To achieve larger power we have planned to include 20 participants per group but we succeed in doing so only for young adults groups.

Before the experiment, all participants answered questionnaires about anxiety and depression -HADS [START_REF] Zigmond | The Hospital Anxiety and Depression Scale[END_REF], mood -PANAS [START_REF] Watson | Development and validation of brief measures of positive and negative affect: the PANAS scales[END_REF], and immersive tendencies -QIT [START_REF] Witmer | Measuring Presence in Virtual Environments: A Presence Questionnaire[END_REF]. A oneway ANOVA was conducted to compare anxiety (HADS-A), depression (HADS-D), positive and negative affect (PANAS-PA and PANAS-NA respectively), and immersive tendencies (IT) between the four experimental groups. No significant difference was found for all of the questionnaires. Moreover, children obtained scores within normal performances in two subtests (Matrix Reasoning and Similarities) of the Weschler Intelligence Scale for preadolescents: WISC V, French version.

Materials and stimuli

Apparatus

We used the HTC Vive headset (first generation) with a spatial resolution of 1080 x 1200 pixels per eye, and 90 Hz refresh rate. To run the experiment, the PC we used had the following specifications: an Intel® Core™ i8-7700HQ CPU with 2 x 3.60GHz, DDR 4 2 x 32 GB RAM, and a Geforce® GTX 1080, running Microsoft Windows 10. We used the crossplatform game engine Unity to create the experiment, and sounds were played through the HTC Vive headphones. We implemented the Pupil Labs add-on for eye tracking to our HTC Vive headset in order to measure pupillary dilation. The HTC Vive HMD features an accelerometer, a gyroscope, and laser position sensor, making it possible to track head and controller rotation and position (see Supplementary Material for detailed description of recording and processing of these data). We also used an Arduino system to measure heart rate with an ear clip pulse sensor.

Stimuli

The stimuli used in the experiment were fifty-eight 3D assets (i.e., 3D models of objects or animals, animated or not, with or without sound) from a larger set of 110 assets, previously pre-tested for valence and arousal by 31 students from Lyon 2 University (see supplementary material and Cadet & Chainay [START_REF] Cadet | Memory of virtual experiences: Role of immersion, emotion and sense of presence[END_REF] for more detailed information concerning the stimuli) .

These assets were split into 12 sets of stimuli. Half of these sets were displayed in a virtual Island environment (in one of the following places : a forest, a beach or a desert), with 3 sets of negative stimuli with high arousal (2 sets of 5, and 1 set of 4), and 3 sets of neutral stimuli (2 sets of 5, and 1 set of 4). The other half was included in a virtual City environment (in places representing different coffee shop terraces, 2 sets of 5 negative stimuli with medium arousal, 2 sets of 5 neutral stimuli, and 2 sets of 5 positive stimuli with medium arousal). We have chosen these two environments and the six places in each environment to be able to coherently present each set of the stimuli (i.e., each set in one of the 6 specific places in Island and the same in City environment). The sets of stimuli were created such that the sets including the stimuli of the same valence (i.e., negative, positive or neutral) are of a similar level of valence and arousal.

The mean scores, ranges and standard deviation for valence and arousal for each set are available in the supplementary material in Table 1.

Procedure

Participants were seated in an armchair. After setting up the VR HMD, the headphones, and ear clip pulse sensor, the participants were asked to give the signal when they were ready to start the experiment. We first proceeded with eye-tracking calibration. After this calibration, the experiment settings menu appeared, and a computer-synthetized voice explained the aim of the experiment, then the participants were asked to stay still and calm for one minute in order to calculate their resting heart rate. Following this, and before starting to visit the environments, for the Avatar condition, participants were asked to choose the color of their futuristic suit by pointing it through the controller's raycast and pressing the controller's trigger. As previously reported [START_REF] Waltemate | The Impact of Avatar Personalization and Immersion on Virtual Body Ownership, Presence, and Emotional Response[END_REF], this personalization was supposed to help the illusion of virtual body ownership.

A mirror was placed in front of them for 30s before the selection of the Island or the City environment and they had to move their arms and head in order to see their avatar moving with them. The mirror appeared also for 5 s before visiting each place. The arms and body of the avatar in the first-person view (i.e., like a person could see his/her own body in real life) was visible for the participants throughout the experiment, hand tracking was carried out by the two controllers, but there was no leg tracking(participants were asked to move their legs as little as possible). For the no-Avatar condition, participants were able to see the controllers instead of their hands in the virtual experience. They started to visit environments straight after recording the resting heart rate.

Irrespective of the Avatar condition, participants were asked to complete a visit of two environments: City and Island, each containing six places. The order in which the environments and places within each environment were visited was selected by the participants. When the participants had selected an environment, they had to visit the six places of the currently explored environment before visiting the second one (see Figure 1). In each place, stimuli were presented one by one during 5s each and with 7s of interval between stimuli, in random order, and always within 180° of the space in front of them. The total time of presentation per place was 60s except 2 places in Island environment (48s). A spatialized sound was played before the stimulus in order to help participants to located it, and they were asked to point out each stimulus before it disappeared.

Insert Figure 1 here At the end of the visit of each place, the participants answered questions displayed in the HMD and played using synthetized speech. First, they rated, on a 9-point illustrated scale, the level of valence (from "very unpleasant" to "very pleasant"), and arousal (from "weakly arousing" to "very arousing") (SAM test, [START_REF] Bradley | Measuring emotion: the Self-Assessment Manikin and the Semantic Differential[END_REF]) in respect of the visited place. Then, they answered the following questions, on a 7-point scale (from "not at all" to "absolutely"): How present did you feel in the environment? (general sense of presence); How much of an impression did you have of being able to visit the environment? (sense of presence relative to their ability to visit the place); How much capable did you feel of touching things? (sense of presence relative to the ability to touch things); How focused did you feel on the task that you were asked to do? (attentional focus), and; How motivated did you feel to do the task that you were asked to do? (motivation). These five questions were selected because they fit with the subscales usually included in extended questionnaires measuring presence (e.g., ITC-SOPI, [START_REF] Lessiter | A Cross-Media Presence Questionnaire: The ITC-Sense of Presence Inventory[END_REF]). For the Avatar condition, the mirror appeared briefly (5s) after the questionnaire, and before selecting a new place to visit; for the no-Avatar condition, nothing occurred during these 5s. To select environments, places and answers in the questionnaires, the controller's raycast pointing accompanied with trigger pression was used for both avatar and no-Avatar conditions.

Once the participant had visited the six places of the first environment, the HMD was removed and they were asked to respond to a French version of the ITC-SOPI questionnaire (composed by four factors: spatial presence, engagement, ecological validity / naturalness, negative effects), and then to recall all stimuli that they had seen in the places just seen ((in Island (28 stimuli) or City (30 stimuli) environment depending on the order of visit that participant has chosen)). Responses were written by the investigator. At the end of the recall, the investigator could ask for details about an ambiguous response; if the participant's answers did not allow the item recalled to be identified, it was counted as an incorrect response. Then, the same procedure was repeated for the remaining environment.

Statistical analysis

Separate statistical analyses of ANOVA for the Island and City environments were conducted. We performed the 2 x 2 x 2 ANOVAs for Island and 3 x 2 x 2 ANOVAs ((Emotion)x(Avatar)x(Age)) with Emotion (for Island -negative vs. neutral; for City -positive vs. negative vs. neutral) as within-subject factor, and Avatar (avatar vs. no-avatar) and Age (Adults vs. Preadolescents) as between-subject factors. If necessary, to better understand the significant effects, planned comparisons were performed. These analysis were conducted on scores of correct free recalls (item memory for virtual stimuli), on the mean sense of presence (score based on the responses to five questions for each place), and emotional valence and arousal (SAM test). Each mean was obtained using scores for places presenting stimuli of the same emotional valence (Island -negative and neutral; City -positive, negative, and neutral).

In addition, an ANOVA was conducted on the four ITC-SOPI factors with Environment (Island vs. City) as within-subject factor, Avatar (avatar vs no-avatar), and Age (Adults vs Preadolescents) as between-subject factors.

Preliminary analyses were performed to check for sphericity (Mauchly's test), and homogeneity of variances (Levene's test). If a violation was found, corrected scores (Greenhouse-Geisser) were used.

The correlation and regression analyses were also performed to determine which of the subjective and objective measures of emotion and presence are linked to the recall performance and could explain this performance.

The results are presented in the following order: (1) recall performance, (2) self-reported measures for sense of presence, arousal, and valence, and (3) correlations and regression analysis.

Results

First, we resume in Table 1 the results of the ANOVAs analysis in the Island and City environment with Emotion as within-subject factor, and with Age and Avatar as betweensubject factors.

Insert Table 1 here

Recall performances

Island

A significant effect of Emotion (F(1, 68) = 87.13, p < .001, partial ŋ 2 = 0.56) was observed with higher recall of negative stimuli (M = 8.71, SE = 0.25) than of neutral stimuli (M = 6.18, SE = 0.18). The effect of Age was also significant (F(1, 68) = 11.54, p < .001, partial ŋ 2 = 0.15), with higher recall for adults (M = 8.04, SE = 0.23) than for preadolescents (M = 6.86, SE = 0.26). Interaction between Age, and Avatar was significant (F(1, 68) = 7.46, p = .008, partial ŋ 2 = 0.10) (see Figure 2). The planned comparison revealed that for the no-avatar condition, the recall was higher for adults (M = 8.38, SE = 0.34) than preadolescents (M = 6.25, SE = 0.36), but in the avatar condition, the difference between adults (M = 7.70, SE = 0.33) and preadolescents (M = 7.46, SE = 0.37) was not significant. There were no other significant effects.

Insert Figure 2 here

City

A significant effect of Emotion (F(2, 136) = 27.02, p < .001, partial ŋ 2 = 0.28) was observed, with higher recall of negative stimuli (M = 5.42, SE = 0.19) and positive stimuli (M = 5.44, SE = 0.21) than of neutral stimuli (M = 3.80, SE = 0.20; respectively t(136) = 6.32, p < .001 and t(136) = 6.40, p < .001); no difference was observed between the positive and the negative stimuli. The effect of Age was also significant (F(1, 68) = 12.33, p < .001, partial ŋ 2 = 0.15), with higher recall for adults (M = 5.36, SE = 0.18) than for preadolescents (M = 4.42, SE = 0.20). There were no other significant effects.

Self-reported sense of presence -SoP evaluation of places

Island

No significant effects were found.

City

Only the effect of Age was significant (F(1, 68) = 4.87, p = .031, partial ŋ 2 = 0.07), with preadolescents (M = 22.20, SE = 0.76) reporting feeling more present in the places than adults (M = 19.80, SE = 0.73).

Self-reported sense of presence -ITC-SOPI -evaluation of environments

Significant effects of Age were found, independently of the environment, for the Spatial

Self-reported arousal

Island

Only the effect of Age was almost significant (F(1, 68) = 3.41, p = .069, partial ŋ 2 = 0.05), with preadolescents (M = 5.68, SE = 0.27) rating the places as more arousing than adults (M = 5.03, SE = 0.24).

City

A

City

A significant effect of Emotion (F(1. Insert Figure 4 here

Correlations and regression analysis

Correlations

Correlation analyses for all groups, then separately for adults and preadolescents, were performed between the percentage of correct recall and the mean scores of self-reported arousal, valence, sense of presence (SoP) per VE, and the mean per VE of different measures of head movements (standard deviation of the head position on the x-, y-, and z-axis, standard deviation of the head rotation on the x-, y-and z-axis, respectively, pitch, yaw and roll), pupillary dilation.

For the record, a higher standard deviation of the head position of rotation means more movements.

Island

Independently of the age-group, Pearson's test showed a significant correlation between recall performance and the valence rating (r = -.134, p = .002), and the standard deviation of the head position on the y-axis (r = .083, p = .039). For the adult group, Pearson's test also showed significant correlation between recall performance and the standard deviation of the head position on the y-axis (r = .148, p = .011), and the z-axis (r = .131, p = .021). For preadolescents, there were no significant correlations.

City

Independently of the age-group, Pearson's test showed a significant correlation between recall performance and the valence score (r = -.131, p = .003), and the standard deviation of the head position on the x-axis (r = -.083, p = .040). For the adult group, Pearson's test showed a significant correlation between recall performance and the arousal score (r = .115, p = .038), the valence score (r = -.191, p = .002), the standard deviation of the head position on the y-axis (r = -.125, p = .027), and the BPM (r = .123, p = .029). For preadolescents, there were no significant correlations.

Regression analysis

First, we report the correlations between different predictors (Arousal and Valence ratings, sense of presence, standard deviation of the head position on the x-, y-, and z-axis, standard deviation of pitch, yaw, and roll, pupillary dilation, heart rate (BPM), and heart rate acceleration (Acc_BPM)), and, second, the regression analysis.

Insert Table 2 here

Regression analysis

The stepwise regression analysis was performed separately for the Island and City environments (see Table 3). For each environment, we first included data for all participants, and, second, we analyzed data for each group (adults and preadolescents) separately. The percentage of correct Recall per VE was entered in the models as a dependent variable, and the scores in respect of Arousal and Valence ratings, sense of presence, standard deviation of head position on the x-, y-, and z-axis, standard deviation of pitch, yaw, and roll, pupillary dilation, heart rate (BPM), and heart rate acceleration (Acc_BPM) as predictors.

Insert Table 3 here

Discussion

In the present study we aimed, first of all, to investigate the respective roles of avatar incarnation, emotional responses, and sense of presence in preadolescents' and adults' memory.

In addition, we aimed to examine the possible effects of an avatar incarnation, the emotional nature of the VR environment, and age on emotional responses and sense of presence. To interpret and discuss the effects of the above-mentioned factors on memory performances more easily, we will first discuss our results regarding our second objective.

Modulation of sense of presence: effects of avatar and age

Presence was assessed by means of a short questionnaire answered after each place visited in both environments (City and Island), and by means of a global questionnaire -ITC-SOPI [START_REF] Lessiter | A Cross-Media Presence Questionnaire: The ITC-Sense of Presence Inventory[END_REF], assessed after the visit to each environment. Contrary to our hypothesis, sense of presence was not impacted by the incarnation of an avatar either in adults or preadolescents.

Interestingly, Tuena et al. [START_REF] Tuena | How virtual embodiment affects episodic memory functioning: A proof-of-concept study[END_REF] compared three conditions of embodiment in terms of sense of presence, and showed that full embodiment (full-body avatar fully controlled by the participant), and medium embodiment (full-body avatar with body movements controlled by the participant, and navigation executed by the investigator) created more presence than low embodiment (no avatar, and navigation controlled by the investigator). No difference was observed between full and medium embodiment. The authors explained this last result by pointing out the crucial role of motor control in presence. In these two conditions, body movements were controlled by the participant, allowing them to have a stable action-intention continuum. These results are in accordance with the theoretical model of Triberti and Riva [START_REF] Triberti | Being Present in Action: A Theoretical Model About the "Interlocking" Between Intentions and Environmental Affordances[END_REF] that emphasizes a close relationship between presence, intention, and interaction in VR. In our study, in the no-avatar condition, the two visible controllers in their current position were used to select responses in emotional and presence questionnaires at the end of each visit. Thus, it is possible that it provided sufficient motor control, and interaction with the virtual environment, and thus generated a stable and predictable action-intention continuum. Our findings complete the research by Tuena et al. [START_REF] Tuena | How virtual embodiment affects episodic memory functioning: A proof-of-concept study[END_REF], and Triberti and Riva [START_REF] Triberti | Being Present in Action: A Theoretical Model About the "Interlocking" Between Intentions and Environmental Affordances[END_REF], by confirming the critical role of action in presence among adults, but also among preadolescents even without an avatar embodiment.

With regard to the impact of age on presence, globally, preadolescents reported a more heightened sense of presence than adults. More specifically, on the global measurement (ITC-SOPI), this applied to spatial presence and engagement dimensions. These results are, at least partially, in line with the results of Baumgartner et al. [START_REF] Baumgartner | Neural correlate of spatial presence in an arousing and noninteractive virtual reality: an EEG and psychophysiology study[END_REF] showing a more heightened sense of spatial presence in children and preadolescents than older adolescents during a realistic rollercoaster ride projected on a wall. According to the authors, this could be due to ongoing maturation of the prefrontal cortex in children, who are, consequently, less able to control and monitor spatial sense of presence. Contrary to Sharar et al. [START_REF] Sharar | Factors Influencing the Efficacy of Virtual Reality Distraction Analgesia During Postburn Physical Therapy: Preliminary Results from 3 Ongoing Studies[END_REF] results, the preadolescents in our study rated the realness of the virtual environment at the same level as adults. These authors suggested that participants aged less than 18 years old rated VR environments as more real, because they are more exposed to video games or other virtual experiences than older adults.

In our study, adults group aged between 18 and 28 years, thus is it possible that our population of adults was equally exposed to video games than our preadolescents group. Concerning the negative effects, no differences were observed between adults and preadolescents ratings. Our data are consistent with a recent study by Tychsen and Foeller [START_REF] Tychsen | Effects of Immersive Virtual Reality Headset Viewing on Young Children: Visuomotor Function, Postural Stability, and Motion Sickness[END_REF] on children (4 to 10 years),

showing good tolerance of 3D virtual reality games, without negative effects, in terms of visuomotor function, postural stability, or motion sickness. Our study points out that all dimensions of presence might not be affected to the same extent by age, but more studies are necessary to explore each of these dimensions specifically.

Interestingly, and somewhat surprisingly, on more fractionated measurements (assessed after visiting each place), the effect of age on sense of presence was observed only for one of the two environments: the City. This might be explained by the multiple differences between the Island and the City environments, such as, familiarity with the environment, level of arousal and valence of the presented stimuli, the fact that the stimuli were animated or/and manufactured or not. For example, authors [START_REF] Baka | An EEG-Based Evaluation for Comparing the Sense of Presence between Virtual and Physical Environments[END_REF] demonstrated that young adults placed in a realistic and more familiar virtual classroom felt more present than those placed in a nonrealistic and less familiar classroom. Emotional content of the VR is known to enhance presence [START_REF] Baños | Immersion and Emotion: Their Impact on the Sense of Presence[END_REF][START_REF] Riva | Affective Interactions Using Virtual Reality: The Link between Presence and Emotions[END_REF]; however, younger people might respond differently to emotional stimuli than adults, especially to positive valence [START_REF] Cordon | Children's and adults' memory for emotional pictures: Examining age-related patterns using the Developmental Affective Photo System[END_REF][START_REF] Sharp | Children's subjective emotional reactivity to affective pictures: gender differences and their antisocial correlates in an unselected sample of 7-11-year-olds[END_REF][START_REF] Syssau | Children's emotional norms for 600 French words[END_REF][START_REF] Vesker | Perceiving arousal and valence in facial expressions: Differences between children and adults[END_REF]. So, even if we did not observe a direct impact of emotion on presence, it is possible that the differences in the emotional nature of the stimuli presented in the City (less arousing stimuli, inclusion of positive stimuli) and Island environments might partly explain the fact that the effect of age on presence was observed only for the City environment. According to Detenber et al. [START_REF] Detenber | Roll 'em!: The effects of picture motion on emotional responses[END_REF], animation of the stimulus increases arousal.

Thus, as the stimuli presented in the City environment was mostly non-animated, altogether, our results could suggest that the effect of age on sense of presence might depend on the level of arousal of the VR environment. In sum, these different patterns of results between the City and Island environments suggest that the effects of age on sense of presence might depend on several factors (e.g., familiarity or animation), but further studies manipulating these factors specifically are necessary to confirm this suggestion.

Globally, our results suggest a crucial role of sensorimotor interaction in sense of presence in VR, as we did not observe an increased sense of presence with the avatar incarnation compared to the no-avatar condition, which also allowed the participants to interact with the virtual environment on a sensorimotor level. In addition, our results suggest that the impact of age on sense of presence might depend on several factors, as we observed that preadolescents experienced more heightened presence than adults only under certain circumstances, especially in more familiar and less arousing environments.

Emotional responses: effects of age and emotional nature of the stimuli

Emotional responses were assessed with two questions of the SAM [START_REF] Bradley | Measuring emotion: the Self-Assessment Manikin and the Semantic Differential[END_REF] assessing arousal and valence after each place visited. Globally, we observed that places were rated consistently with the emotional nature of the stimuli presented in it. We did not demonstrate any impact of the avatar incarnation on emotional evaluations, probably due to the suggestion mentioned above that sensorimotor inclusion matters more than incarnating a body.

We observed an effect of age on valence and arousal ratings. As regards valence, in the Island environment, regardless of the valence (negative or neutral) of the stimuli presented, preadolescents rated places as more positive than adults. Surprisingly, in the City environment, this tendency was observed only for places with negative stimuli. The opposite results were described by Cordon et al. [START_REF] Cordon | Children's and adults' memory for emotional pictures: Examining age-related patterns using the Developmental Affective Photo System[END_REF], with no difference in terms of valence rating between children and adults for negative stimuli, and children rating positive and neutral pictures more positively than adults. It is possible that, in our study on VR, preadolescents confused the emotion triggered by the virtual environment with the enjoyment of being in a virtual environment, and of using an HMD. Visch et al. [START_REF] Visch | The emotional and cognitive effect of immersion in film viewing[END_REF] described this as artefact emotions. Thus, it is probable that the question concerning valence was particularly confusing for children. They might have overestimated the positivity of places with negative stimuli, because of the peculiar sensation of being near dangerous or disgusting things without any risk. It could be interesting to introduce an additional question about artefact emotion in further studies to investigate this hypothesis more specifically.

As regards the arousal rating, in general, preadolescents rated places as more arousing than adults, except places with negative stimuli in the City environment. This result is consistent with Cordon et al. [START_REF] Cordon | Children's and adults' memory for emotional pictures: Examining age-related patterns using the Developmental Affective Photo System[END_REF], but other authors did not demonstrate this effect [START_REF] Leventon | Development in the neurophysiology of emotion processing and memory in school-age children[END_REF][START_REF] Stenson | Emotion effects on memory from childhood through adulthood: Consistent enhancement and adult gender differences[END_REF]. Cordon et al.

[47] obtained the same findings on arousal ratings of images. Surprisingly, unlike adults, in the City environment, preadolescents rated places with positive, neutral, and negative stimuli as equally arousing. It is possible that, in this environment, where the stimuli were less arousing than in the Island environment in general, preadolescents had more difficulties estimating the level of arousal that they experienced. Leventon at al. [START_REF] Leventon | Development in the neurophysiology of emotion processing and memory in school-age children[END_REF] suggested that between the 5 th -7 th year of life, maturation is not complete, and emotional processes are still developing. Thus, one of the consequences might be that emotional experience is less refined in children, and fine evaluation in terms of arousal is rather difficult for young people even in preadolescents, as observed in our study. Moreover, one limitation of this study is that, for technical reasons, we did not pre-tested arousal and valence for each virtual place (e.g., the desert) as we did for each stimulus. Thus, it is possible that the places themselves along with the presented stimuli could have some influence on the participants' evaluation of valence and arousal.

Taken together, our results suggest that in VR, preadolescents are more sensitive to emotional content than adults, probably because of their emotional system is still maturing. The avatar incarnation did not interfere with emotional evaluation, probably because it did not differ enough from our no-avatar condition in terms of sensorimotor interactions.

Memory performances in VR

Avatar incarnation could enhance EEM in preadolescents

We hypothesized that EEM would be modulated by the avatar incarnation, leading to greater recall of emotional stimuli than non-emotional stimuli in the avatar condition, especially for preadolescents. In general, recall was less effective in preadolescents than adults, but, more importantly, in both groups, we observed better recall for emotional than neutral stimuli in the Island and City environments. These results confirm previous studies that showed EEM in children, preadolescents, and adolescents [START_REF] Leventon | Development in the neurophysiology of emotion processing and memory in school-age children[END_REF][START_REF] Stenson | Emotion effects on memory from childhood through adulthood: Consistent enhancement and adult gender differences[END_REF][START_REF] Massol | Emotional modulation of episodic memory in school-age children and adults: Emotional items and their associated contextual details[END_REF]. Partially in accordance with our hypothesis, though only in the Island environment where we observed an interaction between Avatar and Age, we observed that embodying an avatar allowed preadolescents to perform at the same level as adults. This interaction might be explained by the fact that at the descriptive level avatar embodiment seemed to improve, on average, preadolescents' but not adult's memory, although, as shown previously [START_REF] Tuena | How virtual embodiment affects episodic memory functioning: A proof-of-concept study[END_REF] no significant differences were observed neither for adults nor preadolescents between avatar and no-avatar conditions in planned comparisons. Thus, the preadolescents memory performance in our study was somehow more sensitive to embodiment of avatar than that of young adults, but surprisingly only in one environment, the Island. This difference is difficult to explain, because the statistical analysis (ITC-SOPI) and the inspection of descriptive data (SoP, valence and arousal) for the different measures we used in our study do not indicate that these two environments had clearly different impact on our participants.

However, it is possible that some other factors that we did not control and measure have differentially modulated the impact of avatar on memory performance in Island and City environment, such as familiarity, that is otherwise known to have an impact on memory [START_REF] Sala | Experts' memory superiority for domain-specific random material generalizes across fields of expertise: A meta-analysis[END_REF][START_REF] Coutanche | Influences on memory for naturalistic visual episodes: sleep, familiarity, and traits differentially affect forms of recall[END_REF].

Globally, we demonstrated that incarnation of an avatar during the encoding of stimuli could impact memory differently in adults and preadolescents, but more studies are necessary to explore this effect further.

Predicting memory in VR in adults and preadolescents

Regression analysis were performed to investigate which variables could explain memory performances in VR. Our variables of interest were self-reported measures of sense of presence, arousal, and valence, and physiological measurements of pupillary dilation, heart rate, heart rate acceleration, and movements of the head (standard deviation of position and rotation on the three spatial axes).

The regression analysis revealed valence as a predictor of memory performances for the analysis including all participants, in both environments, Island and City. These results seem to be somehow inconsistent with previous studies on adults [START_REF] Roozendaal | Memory Modulation[END_REF] and children [START_REF] Quas | Arousal at encoding, arousal at retrieval, interviewer support, and children's memory for a mild stressor[END_REF], which attributed memory performances more to arousal than valence. However, the analysis of correlations between predictors showed that, in our study, the arousal and valence ratings of each place were strongly correlated with each other. Moreover, valence explained less than 2% of the data variance for the analysis including all participants. So, although our results suggest that valence of the VR environment is a predictor of memory performance in preadolescents and adults, this suggestion needs further investigations.

The separate analysis according to group, showed that for adults, valence was still a predictor of memory performance, along with SD of head roll in the City environment, but only SD of head position was a predictor in the Island environment. As regards these two measures, they are supposed to reflect the exploration of a virtual environment [START_REF] Li | A Public Database of Immersive VR Videos with Corresponding Ratings of Arousal, Valence, and Correlations between Head Movements and Self Report Measures[END_REF][START_REF] Won | Identifying Anxiety Through Tracked Head Movements in a Virtual Classroom[END_REF]. Thus, it is possible that, by changing their point of view frequently, young adults increased their exploration of the stimulus, leading to richer visual encoding, and indeed better recall performances. For preadolescents, no variables were predictors of memory performance. It is possible that the reduced number of participants in the separate analysis prevented from significant effects from being observed in both environments for preadolescents. In addition, in the case of preadolescents, it has been suggested that there is considerable variability in EEM results due to different methodological aspects [START_REF] Quas | Arousal at encoding, arousal at retrieval, interviewer support, and children's memory for a mild stressor[END_REF]. So, it is possible that, along with the reduced number of participants, the fact that we did not find any predictor for memory performance in preadolescents has multifactorial causes. For example, as discussed previously in section 4.2, the self-evaluation of places in respect of valence and arousal was probably affected by the fact that preadolescents probably misunderstood these evaluations. In addition, the correlation analysis between predictors shows that physiological and self-reported emotional measures were not consistent for preadolescents. Indeed, there were only a few correlations between selfevaluated arousal or valence in respect of places and physiological measures (as an example, heart rate, heart rate acceleration, SD of the yaw were correlated with arousal in the Island environment, but not in the City environment -see supplementary material).

To summarize, even if our study suggests that valence is a predictor of memory performance in both preadolescents and adults, and that, in adults, other factors reflecting environment exploration might also contribute to memory performance. In fact, the part of the data variance which is explained by each of our models (global and separate analysis) is less than 6.5%. For reference, Falk and Miller [START_REF] Falk | A primer for soft modeling[END_REF], state that a model regarding human data should strive for a minimum of 10%. Moreover, our interpretation has to be treated with caution, as we observed many correlations between our different predictors. Interestingly, these correlations are consistent with the literature. For example, valence, arousal, and presence correlated with one another, as previously reported by several other studies [START_REF] Baños | Immersion and Emotion: Their Impact on the Sense of Presence[END_REF][START_REF] Makowski | Being there" and remembering it: Presence improves memory encoding[END_REF][START_REF] Riva | Affective Interactions Using Virtual Reality: The Link between Presence and Emotions[END_REF][START_REF] Västfjäll | The Subjective Sense of Presence, Emotion Recognition, and Experienced Emotions in Auditory Virtual Environments[END_REF]. The standard deviation (SD) of pitch was significantly correlated with self-evaluated arousal (only in the Island), valence, and presence, and the SD of yaw was correlated with arousal. Similar correlations were observed by Li et al. [START_REF] Li | A Public Database of Immersive VR Videos with Corresponding Ratings of Arousal, Valence, and Correlations between Head Movements and Self Report Measures[END_REF]. The mean heart rate was correlated with arousal, and the mean acceleration with arousal, and sense of presence in the Island environment, which is consistent with the Rose and Chen [65] study.

Conclusion

In the present study, we showed that EEM can occur in VR in both adults and preadolescents. Interestingly, incarnating an avatar permitted preadolescents to have the similar memory performances than adults. We found that self-evaluated valence in respect of virtual places predicted memory performance, probably more consistently in adults than preadolescents, and that head movements were also involved in adults. This could have practical applications in the field of professional training or learning, by including these measures in protocols. Interestingly these conclusions are not applicable to preadolescents, further studies would be necessary to explore the factors that could predict memory performances in VR for younger population. This study draws particular attention to the fact that results on an adult population are not necessarily extendable to preadolescents.

We also found that valence, arousal, and sense of presence ratings were impacted by age, with more positive and higher ratings of arousal and certain aspects of presence among preadolescents than in adults. So, our results draw attention to the susceptibility young population, even preadolescents, to feeling aroused and present in virtual environments.

Regarding the variability of results on presence evaluations, studies on preadolescents and children should particularly be careful in their choices of questionnaires. Surprisingly, the avatar condition had no effect on these measures, leading us to hypothesize that the ability to interact and be included at a sensorimotor level is more important than the incarnation of a body.

Presence (F( 1 ,

 1 68) = 7.57, p = .008, partial ŋ 2 = 0.10), Engagement (F(1, 68) = 8.11, p = .006, partial ŋ 2 = 0.11), and Naturalness factors ( F(1, 68) = 4.53, p = .037, partial ŋ 2 = 0.06), with higher scores for preadolescents (spatial presence: M = 72.3, SE = 2.18; engagement: M = 59.1, SE = 1.22; Naturalness: M = 18, SE = 0.74) than adults (spatial presence: M = 63.8, SE = 2.18; engagement: M = 54.2, SE = 1.22; Naturalness: M = 15.8, SE = 0.74). For the Negative Effects factors, no significant effect was found.

  significant effect of Emotion (F(1.65, 112.06) = 25.67, p < .001, partial ŋ 2 = 0.27) was observed, with places with negative stimuli (M = 5.12, SE = 0.23) and with positive stimuli (M = 4.26, SE = 0.24) being rated as more arousing than places with neutral stimuli (M = 3.35, SE = 0.23; respectively t(136) = 7.16, p < .001, and t(136) = 3.69, p < .001), and places with negative stimuli being rated as more arousing than those with positive stimuli (t(136) = 3.47, p = .002). The effect of Age was significant (F(1, 68) = 12.39, p < .001, partial ŋ 2 = 0.15), with preadolescents (M = 4.88, SE = 0.27) rating places as more arousing than adults (M = 3.60, SE = 0.24). The interaction between Emotion and Age was also significant (F(1.65, 112.06) = 8.13, p = .001, partial ŋ 2 = 0.11) (see Figure3). The planned comparison revealed that only adults rated places with negative stimuli (M = 5.04, 0.30) and places with positive stimuli (M = 3.46, SE = 0.32) as more arousing than places with neutral stimuli (M = 2.31, SE = 0.31; respectively t(136) = 8.28, p < .001 and t(136) = 3.49, p = .010). Adults also rated places with negative stimuli as more arousing than those with positive stimuli (t(136) = 4.78, p < .001). No difference was observed in preadolescents' ratings between places with negative, neutral, and positive stimuli. Moreover, preadolescents rated places with neutral (M = 4.39, SE = 0.34) and positive stimuli (M = 5.06, SE = 0.35) as more arousing than adults (respectively t(136) = 4.50, p < .001 and t(136) = 3.46, p = .011). No significant difference was observed between adults and preadolescents for places with negative stimuli.A significant effect of Emotion (F(1, 68) = 18.44, p < .001, partial ŋ 2 = 0.21) was observed, with places with neutral stimuli (M = 1.32, SE = 0.16) being rated as more positive than those with negative stimuli (M = 0.34, SE = 0.21). The effect of Age was also significant (F(1, 68) = 28.99, p < .001, partial ŋ 2 = 0.30), with preadolescents rating places as more positive (M = 1.63, SE = 0.22) than adults (M = 0.04, SE = 0.20). No other significant effects were observed.

Figure 1 .

 1 Figure 1. Presentation protocol of the two virtual environments (city and island), and of the six different places in each environment.

Figure 1 .Figure 1 .Figure 1 .

 111 Figure 1. Mean score of Recall performance for the Island environment (min = 0, max = 14), according to Age (adults and preadolescents), and Avatar condition (avatar and no-avatar).

  [START_REF] Detenber | Roll 'em!: The effects of picture motion on emotional responses[END_REF] 102.69) = 62.68, p < .001, partial ŋ 2 = 0.48)

	was observed, with places with positive stimuli (M = 2.37, SE = 0.17) being rated as more
	positive than those with neutral (M = 1.53, SE = 0.17), and negative stimuli (M = -0.02, SE =
	0.22; respectively t(136) = 3.85, p < .001, and t(136) = 11.03, p < .001), and places with neutral
	negative stimuli (M = 1.19, SE = 0.33; t(136) = 3.63, p = .006), but there was no difference
	between places with positive and neutral stimuli (M = 2.03, SE = 0.25) or between negative and
	neutral for preadolescents. Moreover, preadolescents only rated places with negative stimuli as
	more positive than adults (t(136) = 6.40, p < .001).

stimuli being rated as more positive than those with negative stimuli (t(136) = 7.18, p < .001).

The effect of Age was significant (F(1, 68) = 16.03, p < .001, partial ŋ 2 = 0.19), with preadolescents (M = 1.86, SE = 0.21) rating places as more positive than adults (M = 0.73, SE = 0.19). The interaction between Emotion and Age was also significant (F(1.51, 102.69) = 15.90, p < .001, partial ŋ 2 = 0.19) (see Figure

4

). The planned comparison revealed that adults rated places with positive stimuli (M = 2.38, SE = 0.22) and those with neutral stimuli (M = 1.04, SE = 0.22) as more positive than those with negative stimuli (M = -1.23, SE = 0.30; respectively t(136) = 12.48, p < .001, and t(136) = 7.84, p < .001), and places with positive stimuli as more positive than those with neutral stimuli (t(136) = 4.64, p < .001). Preadolescents rated places with positive stimuli (M = 2.36, SE = 0.25) as more positive than those with

Table 1 .

 1 Resume of the results of the ANOVAs analysis in the Island and City environment with Emotion (Islandnegative vs. neutral; City -positive vs. negative vs. neutral) as within-subject factor and Age (Adults vs. Preadolescents), and Avatar (Avatar vs. No-Avatar) as between-subject factors.

	VD	Effects	df	F	p	partial ŋ 2
		ISLAND
	Recall performances	Emotion	68	87.13	< .001	0.56
		Age	68	11.54	< .001	0.15
		Age * Avatar	68	7.46	.008	0.10
	Sense of Presence	NS
	Arousal	Age	68	3.41	.069	0.05
	Valence	Emotion	68	18.44	< .001	0.21
		Age	68	28.99	< .001	0.30
		CITY
	Recall performances	Emotion	136	27.02	< .001	0.28
		Age	68	12.33	< .001	0.15
	Sense of Presence	Age	68	4.87	.031	0.07
	Arousal	Emotion	112.06	25.67	< .001	0.27
		Age	68	12.39	< .001	0.15
		Emotion * Age	112.06	8.13	.001	0.11
	Valence	Emotion	102.69	62.68	< .001	0.48
		Age	68	16.03	< .001	0.19
		Emotion * Age	102.69	15.90	< .001	0.19

Table 2 .

 2 Results of the analysis of correlations between predictors in the Island and City environments for allgroups taken together. Only significant correlations are reported.

		Arousal	Valence	SoP	SD head	SD head	SD head	SD Pitch SD Yaw	SD Roll	Pupillar	BPM	Acc_
		rating	rating		position	position	position				y		BPM
					x	y	z				dilation
							ISLAND
	Arousal
	rating
	Valence	r= -.113
	rating	p= .016
	SoP	r= .420	r= .171
		p< .001	p< .001
	SD head	ns	r= .219	ns
	position x		p< .001
	SD head	ns	r= .120	ms	r= .500
	position y		p= .010		p< .001
	SD head	ns	r= .150	r= .123	r= .589	r= .629
	position z		p= .001	p= .009	p< .001	p< .001
	SD Pitch	r= .120	r= .169	r= .173	r= .151	r= .201	r= .179
		p= .010	p< .001	p< .001	p= .001	p< .001	p< .001
	SD Yaw	r= .095	ns	ns	r= .322	r= .208	r= .132	r= .220
		p= .044			p< .001	p< .001	p= .005	p< .001
	SD Roll	ns	r= .181	r= .120	r= .196	r= .200	r= .155	ns	ns
			p< .001	p= .011	p< .001	p< .001	p= .001
	Pupillary	ns	ns	ns	ns	ns	ns	ns	ns	r= .110
	dilation									p= .019
	BPM	r= .098	ns	ns	ns	r= .139	r= .145	ns	ns	r= .138	ns
		p= .038				p= .003	p= .002			p= .003
	Acc_BPM	r= .121	ns	r= -.128	ns	ns	r= .103	ns	ns	ns	ns	r= .540
		p= .010		p= .003			p= .028					p< .001
							CITY
	Arousal
	rating
	Valence	r= .108
	rating	p= .021
	SoP	r= .372	r= .321
		p< .001	p< .001
	SD head	ns	ns	ns
	position x
	SD head	ns	ns	ns	r= .608
	position y				p< .001
	SD head	ns	r= .146	r= .128	r= .652	r= .787
	position z		p= .002	p= .006	p< .001	p< .001
	SD Pitch	ns	r= .153	r= .113	r= .119	r= .210	r= .213
			p= .001	p= .016	p= .011	p< .001	p< .001
	SD Yaw	r= .095	ns	ns	r= .260	r= .198	r= .156	r= .283
		p= .022			p< .001	p< .001	p< .001	p< .001
	SD Roll	ns	ns	ns	r= .303	r= .286	r= .289	r= .092	r= .234
					p< .001	p< .001	p< .001	p= .049	p< .001
	Pupillary	ns	ns	ns	ns	ns	ns	ns	ns	ns
	dilation
	BPM	ns	ns	ns	ns	ns	ns	ns	ns	r= .100	ns
										p= .033
	Acc_BPM	ns	ns	ns	ns	ns	ns	r= .100	ns	ns	ns	r= .528
								p= .033				p< .001

Table 3 .

 3 Results of stepwise regressions for all device conditions, adults, and preadolescents for the recall task (only significant regressions are reported). Probability of F at .05 for entry, and at .10 for removal. The significance of β values is given in brackets in the tables.

	Variables	Model	Predictors	β, (p)	R 2 adjusted
			ISLAND
			Global
	Percentage Recall	1	Valence	-0.134, (.005)	.016
			ADULTS
	Percentage Recall	1	SD Head Position y	0.148, (.022)	.022
			PREADOLESCENTS
	Percentage Recall		NS
			CITY
			Global
	Percentage Recall	1	Valence	-0.131 (.005)	.017
			ADULTS
	Percentage Recall	1	Valence	-0.191 (.003)	.037
	Percentage Recall	2	Valence	-0.220 (.001)	.063
			SD Roll	0.165 (.011)
			PREADOLESCENTS
	Percentage Recall		NS
	Note.

Acknowledgements

The present study benefited from a Région Auvergne-Rhône-Alpes grant (Direction des Finances 28/06), and from a Direction de la Recherche et des Ecoles Doctorales grant from Lumière University Lyon 2 (DRED n°13-2019). Finally, it also received financial supported from the Institute for Psychology of Lumière University Lyon 2.

We would like to thank the native English-speaking reviewer for proofreading the manuscript; Mr Juloux, the mayor of Clohars-Carnoët for access to the municipal office for enrolling children in the protocol; Ms. Coudurier and Ms. Vincent, teacher and principal at Clément Marot middle school in Lyon, respectively, for their help in recruiting volunteer child subjects for the study, and the company VR Connection for their support. We would also like to express warm thanks to Thomas Fugier for designing the virtual environments, and to Xavier Rocher for his data processing work.


• sparse or 'culminative' tone systems

• in which no more than one pitch pattern is pronounced per word (or longer phonological unit)

• situated within a continuum between non-tonal stress languages and tonal languages without lexical stress (cf. Hyman 2001:1377)

• characteristics

• lexical tone on monosyllabic domains; simple tone inventories (basic tonal categories H & L)

• small number of tonal patterns on polysyllabic domains

• the connection between lexical tone on monosyllabic words and tonal patterns on polysyllabic words is seen as that involving tone spread from its pre-specified location (mostly at the left edge of the prosodic word) rightwards, determining all or most of the tonal melody of the word (e.g. [START_REF] Evans | African' tone in the Sinosphere[END_REF][START_REF] Evans | A bottom-up approach to vowel systems: the case of Yadu Qiang[END_REF] Prosodic Organization [START_REF] Evans | African' tone in the Sinosphere[END_REF] Word Structure

• Lizu morphemes are generally monosyllabic but words are generally disyllabic (over 80% of the collected vocabulary)

• monomorphemic words are for the most part monosyllabic, e.g. / dʒě/ 'water'

• polysyllabic words are mostly composite, e.g. /dʒê-kʰwae/ 'flood' (from /dʒě/ 'water', /kʰwae/ 'be large'), /tôNbu-mu/ 'nose hair'

(from /tôNbu/ 'nose', /mû/ 'animal hair, fur')

• there is also a handful of disyllabic and trisyllabic monomorphemic words, e.g. /mîdzə/ 'hare, rabbit'

Monosyllabic Words

• at monosyllabic level, Lizu distinguishes two lexical tones: rising and high falling in isolation

• the two tones are fairly evenly distributed in the lexicon

• this is a robust opposition, supported by many minimal pairs

• tones are fully independent: any vowel may bear any tone and all consonants, including sonorants and voiced obstruents, can be associated with both tones H + %HL L + %HL Disyllabic Words

• in disyllabic words, three pitch patterns are observed: HH, HL, LH (LL)

• fairly evenly distributed in the lexicon 1. HH: both syllables have a mid-to high-level pitch contour, though the first is slightly higher than the second, and the second syllable has a falling pattern, as in /mîdzə/ 'hare', /səNɡê/ 'lion' (WT seng ge), /mêlŷ/ 'thrush', and /nôNbû/ 'brain' 2. HL: the f0 peak is typically realized before the end of the first syllable, where the pitch starts to fall and continues to fall in the second syllable, as in /mîdzə/ 'pepper', /mêlje/ 'place', and /tôNbu/ 'nose' 3. LL (LH): within the first syllable, there is a mid-level pitch contour with a slight rise. The f0 peak is realized within the second syllable where there is also a clear fall, as in /mutsə/ 'cat', /meljê/ 'wind', and /nɐNbô/ 'deaf person' HH HL LL (LH)

• in contrast to the two lexical tones on monosyllabic words, the threeway contrast on disyllabic words is not supported by minimal sets • many pairwise minimal pairs Tri-and Tetra-syllabic Words

• in tri-and tetra-syllabic words (both monomorphemic and polymorphemic): three common pitch patterns, comparable to the ones over disyllabic domains:

1. HHH: The pitch contour over all syllables remains mid or high level until the last syllable where there is a clear falling pattern, as in /ʃêNbêljê/ 'buttocks' 2. HLL: There is a rising contour on the first syllable, the peak of which varies slightly depending on the length of the word. For a three-syllable word, the peak is typically realized at the beginning of the second syllable, while for a four-syllable word, the peak is further delayed within the second syllable, after which there is a clear falling contour till the end of the word, as in /tɕjɐNtʃʰokɐ/ 'north' (WT byang phyogs)

LLL (LLH):

The f0 peak is realized within the last syllable and the preceding syllables show a mid-level f0 contour, as in /ɲilɐɡû/ 'daytime' HHH HLL LLL (LLH)

• no contrastive minimal sets for the three patterns Tone Derivation Patterns

• dynamic tone combinations

• in compounds, lexical tones undergo sandhi changes

• compounds and phrases beginning with a noun noun-noun, noun-verb, noun-classifier, noun-clitic

• the synchronically productive pattern as attested on syntactic compounds is one in which only the tonal contour of the initial word is retained and realized over the whole compound domain Tone Derivation Patterns

• if the domain-initial monosyllabic root or word has the rising tone, the resulting pattern is generally L(…L)H

• if the domain-initial monosyllabic root or word has the falling tone, the resulting pattern is generally H(…)H

• in domains that begin with a disyllabic root or word, the resulting contours are determined by the tonal contour of the initial disyllabic item

Noun-Noun Compounds Noun-Noun Compounds Summary

• the synchronically productive pattern: only the tonal contour of the initial word is retained and realized over the whole compound domain

• different tonal contrasts on the monosyllabic level (2 lexical tones) and on the level of words of over one syllable (3 contrastive patterns on disyllabic words) • preference in Lizu for syllables being grouped into binary units

• the binary preference is an instance of a strong crosslinguistic bias found in stress systems

• possibility for an analysis of Lizu as combining lexical tone with stress

• an analysis allowing for the contextualization of the phonetic properties of stress in Lizu relative to the broader literature on acoustic correlates of prominence

Stress

• stress (Hyman 2009: 217): a language with stress-accent is one in which there is an indication of word-level metrical structure meeting the following two central criteria A. obligatoriness: every lexical word has at least one syllable marked for the highest degree of metrical prominence (primary stress) B. culminativity: every lexical word has at most one syllable marked for the highest degree of metrical prominence C. stress-bearing unit is the syllable

• stress is a relative property arising from a relationship between two (or more) syllables, put differently, syllables are perceived as stressed only by virtue of their relationship with nonstressed syllables Acoustic Cues for Stress

• in stress languages, such as English, the principle auditory cues that correlate with word stress are duration, intensity, and vowel quality (e.g. Fry, 1958;Bolinger, 1961;[START_REF] Gordon | Metrical structure and stress[END_REF] • stressed syllables tend to have greater intensity, longer duration, and full (nonreduced) vowel quality, compared with nonstressed syllables

• stressed syllables also tend to have higher pitch (fundamental frequency, F0)

Teasing apart Acoustic Cues for Stress and Tone

• the difficulty of teasing the two apart is canonically due to analyzing words in isolation

• Pierrehumbert (2000): "The citation form is reconstructed as the form produced in a specific prosodic context -when the word is both phrase-final and bears the main stress of the phrase."

Pierrehumbert, Janet. 2000. Tonal elements and their alignment. In M. Horne (ed.), Prosody: Theory and Experiment. Studies Presented to Gosta Bruce. Kluwer, Dordrecht, 11-26.

Experiment

• measurement parameters: intensity, duration, and f0 (= common correlates of stress in languages of the world)

• basis: recordings of eight subjects (four female and four male), all of whom were native speakers of Lizu (the /ʃaetɕʰôpae/ dialect) (age 23-70)

• the list of targeted words: 30 disyllabic lexical words, presented to the subjects to read in randomized order in two contexts in isolation, where the word is both phrase-final and bears the main stress of the phrase in a phrasal context

• in each context, the target words were read each four times

• measurements taken (in Praat): mean intensity, mean duration, and fundamental frequency (1/3, 2/3, 3/3) over each vowel in the word

• after analysis, data were subjected to statistical analysis (repeated measures ANOVAs (analyses of variance))

• the three tone patterns were compared in two contexts (isolation, phrase) as a function of (1) average intensity and duration (all speakers), and ( 2) fundamental frequency at the 1/3, 2/3, 3/3 of the syllable (female and male speakers separately)

• post hoc comparisons using the Tukey HSD test, t-tests

• given that stress is a relative property arising from a relationship between two syllables, comparisons for intensity and duration were based on the differences between two syllables in each word (value Syl1 -value Syl2)

Questions

• can we detect phonetic properties of stress in Lizu?

• what is the correlation between stress and tone?

• are the three patterns on disyllabic words really contrastive?

• why do words of two syllables and longer have three tonal patterns, while there are only two lexical tones on monosyllabic words?

• HH > H(…)H, LH > L(L…L)H, ? > H(L…L)L?

Context: Isolation

  • questions: are the three patterns really contrastive? how do the 2 lexical tones on monosyllabic words transform into 3 tonal patterns on words of two syllables and longer? HH > H(…)H, LH > L(L…L)H, ? > H(L…)L? HL Tonal Pattern • lexicalized compounds (high-frequency, disyllabic noun-verb compounds) ɣǒ 'wine' + tɕʰê 'to drink' > ɣô tɕʰe 'to drink wine; wine-drinking' • recent loanwords from Mandarin: kwâtsə 'seeds', SW Mandarin /kwa⁴⁴tsə⁵³/ • tone change in compounds lêmae 'finger' > lemaê je-je 'little finger'kʰêNtsʰae 'remember' > kʰeNtsʰaê pʰaě 'be able to remember' the lexicon: Lizu morphemes (lexical and grammatical) are generally monosyllabic, but words are generally disyllabic

  | wa-wa | wa-wa | … HH: wa-wa | wa-wa | wa-wa | … LL: wa-wa | wa-wa | wa-wa | …

Context: Phrase •

Intensity

In the phrase-initial position, the three tonal patterns differ significantly as a function of MinInt (difference in the intensity between the two syllables) MinInt : F(2, 1032) = 70.41, p < 0.001 Post-hoc Tukey's HSD show that there is a statistically significant difference between the pattern HL&HH, HL&LL, but there is no difference between the patterns HH & LL Context: Phrase •

Duration

In the phrase-initial position, the three tonal patterns differ significantly as a function of MinDur (difference in the duration between the two syllables) MinDur : F(2, 1032) = 13.34, p < 0.001 Post-hoc Tukey's HSD show that there is a statistically significant difference between the pattern HL&HH, HL&LL, but there is no difference between the patterns HH&LL Pitch: Women 1/3: 1st syllable: F(2, 524) = 113.9, p < 0.001; post-hoc Tukey's tests : no difference between HL&HH; other comparisons not significant 2nd syllable: F(2, 522) = 24.3, p < 0.001; no difference between HL/LL; other comparisons not significant 2/3: 1st syllable: F(2, 524) = 139.7, p < 0.001; no difference between HL/HH; other comparisons not significant 2nd syllable: F(2, 523) = 27.91, p < 0.001; no difference between HL/LL; other comparisons not significant 3/3: 1st syllable: F(2, 524) = 148.6, p < 0.001; no difference between HL/HH; other comparisons not significant 2nd syllable: F(2, 520) = 26.03, p < 0.001; no difference between HH/LL; other comparisons not significant Putting it all together

• Lizu has both lexical tone (pitch) and stress (intensity, duration, pitch)

• stress: on the initial syllable in the domain (trochaic)

• the three tone patterns on disyllabic domains are contrastive, however, the contrast is heterogenous, combining two tonal patterns (HH&LL) and one stress-only pattern (HL)

• all words are stressed (+stress), whereas not all words may be specified for tone (+/-tone)

Interaction of Tone & Stress

• + stress, + tone: if the initial, stress-able syllable in the domain is specified for tone (one of the two basic tones on monosyllabic words: H & L), that tone is realized on the stressed syllable and spreads to the right onto the remaining syllables

• synchronically productive tone derivation rules

• + stress, -tone: if the domain-initial syllable is not specified for tone, the (disyllabic) domain receives the default HL pattern (rightedge demarcating trochaic stress; with a maximum reduction in terms of duration of the second syllable)

• lexicalized combinations, loanwords, etc.

Stress:

Tone & Stress in Lizu

• comparative work

• a hybrid system, combining tone and stress: a crosslinguistically unusual type

• separate stress and tone for comparative purposes

• although future consensus might suggest that Lizu is better classified as lacking stress, the present analysis allows for contextualizing their phonetic properties relative to the broader literature on acoustic correlates of prominence http://wals.info/combinations/14A#5/30.449/107.205

Thank you for your attention!