Daisy Yoo
email: dyoo@cc.au.dk

Peter Dalsgaard
email: dalsgaard@cavi.au.dk

Alix Ducros
email: alix.ducros@liris.cnrs.fr

Aurélien Tabard
email: aurelien.tabard@liris.cnrs.fr

Eva Eriksson
email: evae@cc.au.dk

Clemens Nylandsted
email: clemens@cavi.au.dk

Putting Down Roots: Exploring the Placeness of Virtual Collections in Public Libraries

Keywords: Public libraries, place, virtual collections css concepts, Human-centered computing~Human computer interaction (HCI)~Empirical studies

In this pictorial, we investigate how a virtual collection can put down its roots in a physical space and integrate into the local community. We present PLACED, a placecentric digital service that supports participation and community-production of knowledge in library events. We illustrate how PLACED has been deployed and used at a local public library over a six-month period. We examine the community-produced virtual collection that grew out of this library event with a focus on its placeness.

INTRODUCTION

The advances of the Internet, mobile technologies, and digitalization bring the promise of access to information any time, anywhere [START_REF] Costantino | Exploring Participation in the Design of Public Library e-Services[END_REF]. While we have witnessed remarkable benefits from this development, it also means that information is increasingly becoming placeless [START_REF] Odom | Placelessness, spacelessness, and formlessness: experiential qualities of virtual possessions[END_REF]. This poses fundamental challenges for public libraries, which have traditionally occupied a central position in society as the local gateway to knowledge. In response, many libraries are undergoing institutional transformations to redefine their role, integrating and developing a number of public services, such as offering various types of social support and organising a wide range of community events, both educational and recreational [START_REF] Serholt | Opportunities and challenges for technology development and adoption in public libraries[END_REF].

Over the past three years, we have worked on a cross-European research project to explore this transition. We have worked in close collaboration with public libraries to design, develop, and deploy digital services that support libraries and publics in organizing community events, capturing the knowledge created in these events, and storing, curating, and linking this knowledge with the library collection. One of the key insights that has emerged from this work is the value of connecting the digital content to the physical library space, to the local practices that unfold in library events, and to the community members who take part in them. Fostering such connections can anchor technology into places, and thus, develop placeness.

In this pictorial, we examine how a novel digital service has been deployed at a local public library over the course of six months in a weekly series of creative workshops. Specifically, we will illustrate and reflect on the virtual collection that grew out of this library event with a focus on its placeness. Drawing from theories of place [START_REF] Dourish | Re-space-ing place: " place" and" space" ten years on[END_REF][START_REF] Harrison | Re-placeing space: the roles of place and space in collaborative systems[END_REF][START_REF] Tuan | Space and place: The perspective of experience[END_REF] and the concept of genius loci [START_REF] Dalsgaard | Designing for participation in public knowledge institutions[END_REF], we reflect on how the virtual collection was integrated with the physical library space.

RELATED WORK

Historically, public knowledge institutions have served as iconic physical third places in local communities. Thus, Dalsgaard and his colleagues [START_REF] Dalsgaard | Designing for participation in public knowledge institutions[END_REF] have long advocated that design for public knowledge institutions should "respect the sense of place." Dalsgaard and colleagues turned to the notion of genius loci (Latin for "the spirit of the place") to denote the distinctive character or atmosphere of a place. Genius loci can be understood as the amalgamation of shared socio-cultural meanings that are attributed to a specific place by its community members. To support the (co-)construction of the genius loci, Dalsgaard and colleagues called for designers to explore ways of encouraging participation through various forms of community-generated content contribution; so that the library user will not merely consume but give something meanigful to the place.

The PLACED system we present here responds to this call. Furthermore, our work strongly resonates with Reich and Weiser's earlier work [START_REF] Reich | Libraries are more than information: Situational aspects of electronic libraries[END_REF] in which they argued that we need to temper naive enthusiasm for building a completely placeless, universal information system. They argued that public libraries have a major head start in this area to explore community-based information systems that encourage each local community to create its own network culture and identity.

Our work is also inspired by Odom et al's work [START_REF] Odom | Investigating the presence, form and behavior of virtual possessions in the context of a teen bedroom[END_REF][START_REF] Odom | Placelessness, spacelessness, and formlessness: experiential qualities of virtual possessions[END_REF] on virtual possessions. Yet, our work differs from Odom et al's work in that we deal with public rather than private collections. We explore publicness by drawing on research into commons [START_REF] Ostrom | Governing the commons: The evolution of institutions for collective action[END_REF], which examines shared resources and social practices that evolve around digital media. Whereas considerable attention has been directed at the legal aspects of digital commons [START_REF] Yochai | Commons and Growth: The Essential Role of Open Commons in Market Economies[END_REF][START_REF] Lessig | Reclaiming a commons[END_REF], in recent years, there has been a growing interest in developing design strategies for commoning, or commons design, including [START_REF] Balestrini | A City in Common: A Framework to Orchestrate Large-scale Citizen Engagement around Urban Issues[END_REF] and [START_REF] Marttila | Towards commons design in participatory design[END_REF]. We build on commons design by exploring the ways in which community-produced virtual collections can be linked with the traditional library collection, and thus become an ever-growing part of the urban community's shared resources (i.e., commons).

About the Creative Workshop Series Event Organizer's Digital Toolbox Library Calendar

Event organizers must provide a title, thumbnail image and a brief description of the activity for public announcement.

Pinterest "It's a very, very creative site [...] I use it actually to collect ideas for myself and for the workshop, but I've never contributed to it."

To gain deep, situated insights into designing place-centric interactive technologies for library events, we worked in close collaboration with the organizer of the Creative Workshop series at a major library in Denmark. The goal of this library event is to introduce participants to different creative practices so that people can learn new skills, get inspired, and connect with each other through making. The Creative Workshop is offered twice a week. The event organizer chooses a different theme each week, sometimes taking inspiration from an ongoing local events (e.g., music festivals). Typically participants join the event as a family with young children. People can drop in anytime during the workshop session (1.5 hours) without registration.

While many library events are one-off events, the Creative Workshop series happened all year long, thus granting us the time needed to build a strong relationship with the workshop organizer for engaging in a Participatory Design process. We conducted a series of field observations, contextual inquiries, focus groups, and co-design workshops to understand how events are organized in public libraries.

The Creative Wrokshop organizer identified Pinterest, Instagram, and the camera on her personal mobile phone as the most frequently used tools. In addition, the organizer needed to regularly update new event information on the library calendar.

Mobile Phone Gallery

"I take a lot of photos, and actually they just end up in my phone most of them, but I have to do it because otherwise I forget all the good ideas."

Instagram "I just follow a lot of creative people and just put up a lot of things. [...] It's my kind of portfolio of creativity."

Placing the Creative Workshop in the Library

"But a lot of people don't know! [...] Actually we have this blindness of information. Information blindness."

Here we summarize key areas and digital technologies that contribute to the genius loci of our research site. In particular, the Creative Workshop series took place in the Children's Lab located on the second floor within the kids zone. Children are allowed to play and make noise in this area. Note that most public displays are located in passageways, and despite continuous streams of information, they are scarcely noticed by most visitors.

A large screen near the main entrance displays short video clips promoting the main library events as well as Instagram posts automatically tracked through the hashtag.

Each week, the workshop organizer put out an A-frame chalkboard sign in front of the Children's Lab to announce a new theme.

A public display on the second floor is dedicated to child-friendly events. Eight event posters appear in rotation, including the Creativity Workshop series.

The workshop organizer occasionally posts on her personal Instagram account about the Creative Workshop, knowing that it will show up on the library's main public display.

About the PL ACED DOCUMENTATION MODULE VISUALIZATION MODULE

To support the Creative Workshop series, we developed a community-based information sytem. The PLACED system consists of two modules: a documentation module designed for personal mobile devices, and a visualization module for interactive public displays. Both modules are connected to the library calendar and collection APIs.

The visualization module offers a list of past, ongoing, and upcoming events. Photos and videos captured with the documentation module show up within seconds on the public display. Users can interact with the content through touch (e.g., zoom for images and playback for video files).

Publics can use the system without registration via shared community account.

Registered library staff can access settings and moderate content via librarian account.

Users can upload photos and videos, create polls, or post Q&As.

Inspiration channel allows users to share creative ideas that are not directly tied to a specific event theme.

Each event has a virtual bookself linked to the library database. The system offers smart book suggestions, which users can filter with a Tinderlike swipe interaction.

Deployment : july -december, 2019

We deployed PLACED "in the wild" from July to December 2019. We installed a 55-inch interactive public display in an open sitting area near the Children's Lab. Participants often hanged out in this area before the event as they waited for the Creative Worshop to open as well as after the event to refresh. Although the visualisation module was located outside, it was still visible to the event participants from within the Children's Lab through to glass walls. The display was slightly tilted and positioned low enough for young children to reach and interact with. To the left, a non-interactive public display silently streamed animation films. To the right, bookshelves were lined up in the children's literature section.

In what follows, we present four key insights for placing community-produced virtual collections in public libraries. The insights are derived from the extensive field observations during the six-month deployment period as well as the formal and intermal interviews conducted with the Creative Workshop organizer, who we refer to as Krea-Marie hereafter. PLACED inspired the event organizer to reconfigure her work space. The glue-gun table was tranformed into a bookshelf, bringing the virtual book collection into life. Placing the PLACED 1. connecting to the space "I have needed some ways to connect [the Creative Workshop] to the library, with the books and all the knowledge that is around us," Krea-Marie said. PLACED acted as a mediating aretifact [START_REF] Zander | Collaborative process change by inscription: A contested terrain for interaction designers[END_REF], offering new ways to link the event (e.g., themes, crafts) with the library space and resources (e.g., books, exhibitions). In particular, the event organizer used PLACED to create a quiz related to an ongoing exhibition in the children's literature section. Each week, the event organizer created an event bookshelf, both digitally and physically.

Placing the PLACED 2. connecting people PLACED connected people by providing playful objects for families to collectively work on, reflect on, and talk about. PLACED brought families closer together, even the remote members, to understand each other better. , it could be a part of this big exhibition, we talked about it, it was very interesting. And when they got home, they uploaded the photo of all their stuff. So that was my hope that more families will do that and work more with the [creative] themes when they got home." PLACED connected parents and children in the process of making videos. For example, a father held up the spaceship in front of the camera while his kid was still busy packing up his stuff. A few minutes later, the kid came out to check out the video and pointed out that it's not how it worked. The kid explained to the father how the spaceship would launch into space to dock to the station on the other side. The father gained a better understanding of what the kid had in mind. Together, they collaborated to document a new video from a shared understanding. Genius loci is about social meaning-making [START_REF] Dalsgaard | Designing for participation in public knowledge institutions[END_REF]. Likewise, Dourish defined place as "the ways in which settings acquire recognizable and persistent social meaning in the course of interaction" [START_REF] Dourish | Re-space-ing place: " place" and" space" ten years on[END_REF]. Importantly, it takes time for such social meanings to emerge and grow. The prolonged deployment period allowed us to observe the ways in which people gradually came to value PLACED. Notably, in October 2019, roughly three months into the deployment, we have begun to see people coming back to find their photos and videos from past events, appreciating their memoires preserved on PLACED. As such, PLACED provided a foundation on which to build a collective memory.

On October 23, a boy asked Krea-Marie if she can help him find his "Spaceman Mr. Mustache" that he had made before. When the photo appeared, the boy gave a huge hug to Spaceman Mr. Mustache over the screen with a big smile on his face.

In this pictorial, we explored how communityproduced virtual collections can reinforce the genius loci in public libraries. We did so by visually contextualizing how PLACED had been deployed and used in a particular library event over a sixmonth period, reflecting on how PLACED gave rise to new forms of participation and communityproduction of knowledge in library events, and how the local community came to value their virtual collection over time.

Specifically, we highlighted four ways in which PLACED contributed to the (co-)construction of genius loci in the public library: (i) by inspiring more active use of the physical library space and resources (e.g., rearrangement of bookshelves), (ii) by encouraging social interactions (e.g., stimulating intergenerational dialogue), (iii) by capturing local colors, and (iv) by building a collective memory of the event over time.

Importantly, PLACED acted as a mediating artefact not only for configuring the relationship between the virtual collection and the library collection, but also for grounding the event itself more firmly in the library space. The Creative Workshop organizer reflected: "Personally, I learned to use the books more in the workshops and how to [do so], which I needed a way into before. I became more aware of using all the knowledge in the house more [by participating in this design project]. […] It makes so much sense that we can connect these things [that we make in the Creative Workshops] with books." While knowledge is becoming increasingly digitized and placeless, we hope that our work and insights will inspire more designers to explore place-centric technologies, particularly, in support of strengthening the genius loci in public libraries and working with local communities.

Reflections : placing the virtual collection in public libraries

Placeless Virtual Collection

Popular social media such as Pinterst provide global platforms that are easy to access, collect, and share ideas. However, the content often lacks sense of place, resulting in a collage of rootless things.

Placed Virtual Collection

"It's fun for me to use the pictures from stuff made here instead of just finding [from] everywhere else."

The creative workshop organizer found it highly relevant and meaningful to engage with, and build upon, locally-sourced materials. It helped to strengthen ties between the event and the library space.

Co-construction of Genius Loci

The virtual collection reflected the atmosphere of the place (e.g., local events, nature, traditions). In turn, the physical library space was reconfigured by the virtual collection.

1 ) 3 )

 13 photos

2 "

 2 FAIRY TALES. Explore the exhibition and see if you can guess what fairy tales children have built in the Creative Workshop. Find the answer on the screen across from the Children's Lab."

2 ""PLACED 4 .

 24 [People told me that] it's fun to look at their own pieces on the big screen, it's like they are famous because what they made is on the big screen. Also, I have noticed when there isn't a workshop and people are just walking around finding books, they also go by the screen to show grandparents or parents or siblings what they had made earlier." -Krea-Marie in the exit interview Beach Art July 30 -31, 2019 "Summer has come to Denmark, which means trips to the beach. The beach is full of fun, beautiful stones and shells [...] So come and join us to give stones and shells new life." Summer is over and autumn has come. [...] KREA-Marie has been in the woods collecting leaves, twigs and cones. This week we will see what nature's materials can become."Placing the PLACED 3. capturing local colorDigital archives can function as rich resources for identity construction and presentation[START_REF] Kaye | To have and to hold: exploring the personal archive[END_REF][START_REF] Odom | Investigating the presence, form and behavior of virtual possessions in the context of a teen bedroom[END_REF]. PLACED supported the Creative Workshop to construct a local identity by capturing local colors. The event organizer selected themes reflecting the seasonal events and traditions, and often used materials collected from nature. In turn, the PLACED community gallery brought out the rich color tones, cultural and natural temporalities of the local community. growing with time

ACKNOWLEDGMENTS

Thanks to KREA-Marie from Dokk1, and all who took part in this study. We are grateful for the technical support provided by CAVI, especially Janus Bager Kristensen and Rolf Bagge. The work is funded by Vinnova 2017-01553, Innovation Fund Denmark 5123-00007B, Agence Nationale de la Recherche ANR-16-ENSF-0004, and JPI Urban Europe EU 693443.


Newmar Pinto Marliére

Marcelo Gustavo Lorenzo

Luis Eduardo Martínez Villegas

Alessandra Aparecida Guarneri
email: alessandra.guarneri@fiocruz.br

Co-existing locomotory activity and gene expression profiles in a kissing-bug vector of Chagas disease

Keywords: Rhodnius prolixus, locomotory activity, gene expression, foraging gene, Chagas disease

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

INTRODUCTION

Locomotory activity is a complex behavioral trait which has been associated to diverse contexts such as foraging, the search for sexual partners or for appropriate hiding places [START_REF] Franco | The underlying genetics of drosophila circadian behaviors[END_REF][START_REF] Guerenstein | Host-seeking: how triatomines acquire and make use of information to find blood[END_REF][START_REF] Kaun | cGMP-dependent protein kinase: linking foraging to energy homeostasis[END_REF][START_REF] Kohsaka | Neural circuits underlying fly larval locomotion[END_REF][START_REF] Takken | Odor-mediated behavior of Afrotropical malaria mosquitoes[END_REF]. Insect locomotion can be modulated by diverse factors, as for example, genetic and phenotypic diversity and the way individuals interact in a community [START_REF] Hughes | Ecological consequences of genetic diversity[END_REF], as well as age, sex, nutritional status, and circadian clocks [START_REF] Cascallares | Role of the circadian clock in the statistics of locomotor activity in Drosophila[END_REF][START_REF] Pompanon | Physiological and genetic factors as sources of variation in locomotion and activity rhythm in a parasitoid wasp ( Trichogramma brassicae )[END_REF] . In addition, environmental conditions such as light, temperature and relative humidity will also impact insect locomotory activity profiles [START_REF] Buchan | Effect of temperature and different sex ratios on physical activity and life span in the adult housefly, Musca domestica[END_REF]Shou et al., 2013). Individual variation will be induced by these different endogenous or exogenous factors that affect the behavioral output through neural or neuroendocrine mechanisms [START_REF] Zupanc | Towards a cellular understanding of motivation: structural reorganization and biochemical switching as key mechanisms of behavioral plasticity[END_REF]. In this scenario, the use of multivariate analyses to probe relations of parameters such as physiology, environment and development on locomotory patterns shown by animals becomes an interesting tool to address the intrinsic complexity hidden behind locomotory datasets (A š monaitè et al., 2016;Thorpe and Crompton 2006;[START_REF] Yamamoto | Exhaustive Multi-Parametric Assessment of the Behavioral Array of Daily Activities of Mice Using Cluster and Factor Analysis[END_REF].

Triatomines are hematophagous insects, which in addition to taking blood from vertebrates, act as vectors of Trypanosoma cruzi. This parasite is the etiological agent of Chagas disease, a neglected health condition that currently affects 5-7 million people, particularly in Latin America (WHO, 2017). These nocturnal insects remain hidden in protected shelters during the day as a consequence of their intense thigmotaxis and negative phototaxis [START_REF] Mota | Lack of segregation between two species of Chagas disease vectors[END_REF][START_REF] Reisenman | Circadian control of photonegative sensitivity in the haematophagous bug Triatoma infestans[END_REF][START_REF] Ward | Behavioural responses of the haematophagous bug Triatoma infestans (Klug) (Hemiptera: Reduviidae) to visual stimuli[END_REF]. During dusk, with the decrease in light intensity, triatomines eventually start a period of spontaneous locomotion, leaving their shelters to search for hosts and then, a few hours before and during dawn they return to the safety of the refuge [START_REF] Ferreira | Activity and shelter-related behaviour in Rhodnius prolixus : the role of host odours[END_REF][START_REF] Lorenzo | Activity pattern in relation to refuge exploitation and feeding in Triatoma infestans (Hemiptera: Reduviidae)[END_REF]. The intensity of the activity of triatomines depends on factors such as nutritional status [START_REF] Bodin | Temporal modulation and adaptive control of the behavioural response to odours in Rhodnius prolixus[END_REF][START_REF] Guarneri | The effect of temperature on the behaviour and development of Triatoma brasiliensis[END_REF][START_REF] Lorenzo | Activity pattern in relation to refuge exploitation and feeding in Triatoma infestans (Hemiptera: Reduviidae)[END_REF] and maturation [START_REF] Bodin | State-dependency of host-seeking in Rhodnius prolixus : The post-ecdysis time[END_REF]. The patterns of this spontaneous locomotory activity, as well as their regulation by endogenous clocks, were well characterized in Triatoma infestans [START_REF] Lazzari | Circadian organization of locomotion activity in the haematophagous bug Triatoma infestans[END_REF]. The daily patterns of locomotory activity of triatomines exhibit a bimodal profile, with an initial peak during the first hours of the scotophase and a second and narrower one that starts preceding the photophase [START_REF] Guarneri | The effect of temperature on the behaviour and development of Triatoma brasiliensis[END_REF][START_REF] Lazzari | Circadian organization of locomotion activity in the haematophagous bug Triatoma infestans[END_REF][START_REF] Lorenzo | Activity pattern in relation to refuge exploitation and feeding in Triatoma infestans (Hemiptera: Reduviidae)[END_REF][START_REF] Marliére | Trypanosomes modify the behavior of their insect hosts: effects on locomotion and on the expression of a related gene[END_REF][START_REF] Pavan | Rhodnius prolixus and R. robustus (Hemiptera: Reduviidae) nymphs show different locomotor patterns on an automated recording system[END_REF]. Previous studies on triatomine locomotory activity have focused their analyses on average tendencies. We suggest here that the factors underlying the inherent variability of triatomine locomotion deserve attention, as they may reveal relevant biological features affecting bug performance under different predation risk scenarios.

Foraging ( for ) is an essential gene with pleiotropic effects on development and behavior (Hofmann, 2005). It encodes a cGMP-dependent protein kinase (PKG) that is found in diverse groups of organisms from Paramecia to humans and presents very conserved functions (Feil et al., 2005). In invertebrates, the foraging gene has been associated with the modulation of food-related behavior in different groups such as Drosophila [START_REF] De Belle | Genetic localization of foraging (for): a major gene for larval behavior in Drosophila melanogaster[END_REF][START_REF] Osborne | Natural behavior polymorphism due to a cGMP-dependent protein kinase of Drosophila[END_REF][START_REF] Pereira | Mutations in the larval foraging gene affect adult locomotory behavior after feeding in Drosophila melanogaster[END_REF], honey bees [START_REF] Ben-Shahar | The foraging gene, behavioral plasticity, and honeybee division of labor[END_REF], ants [START_REF] Ingram | Taskspecific expression of the foraging gene in harvester ants[END_REF], nematodes [START_REF] Fujiwara | Regulation of body size and behavioral state of C. elegans by sensory perception and the EGL-4 cGMP-dependent protein kinase[END_REF], social wasps [START_REF] Tobback | Cloning and expression of PKG, a candidate foraging regulating gene in Vespula vulgaris[END_REF] and locusts (Luccas et al., 2010). We have previously shown that the levels of locomotory activity of Rhodnius prolixus are altered by trypanosome infection [START_REF] Marliére | Trypanosomes modify the behavior of their insect hosts: effects on locomotion and on the expression of a related gene[END_REF]. These changes coincided with an altered expression of Rpfor , the foraging gene orthologue of this species, suggesting that it participates in the modulation of triatomine activity.

In the present study, we characterized the distribution of locomotory events of starved 5 th instar nymphs of R. prolixus along a 24h interval. Then, we tested whether bug locomotory profiles would clusterize evincing differing activity profiles. We subsequently hypothesized that differing locomotory profiles correlate with Rpfor expression. Furthermore, we decided to explore whether nymph sex would affect bug locomotory profiles as seen for adult R. prolixus [START_REF] Pontes | Female odours promote the activation of sheltered kissing bug Rhodnius prolixus males and modulate their orientation[END_REF]. Finally, we addressed whether these variables also affected the cumulative locomotion observed for each individual.

METHODS

Insects

Rhodnius prolixus specimens used in our study came from a colony derived from insects collected in Honduras around 1990.

Locomotory activity

The locomotory activity of nymphs was evaluated by means of an automatic actometric system previously developed by our group (for details see [START_REF] Marliére | Trypanosomes modify the behavior of their insect hosts: effects on locomotion and on the expression of a related gene[END_REF]. Briefly, the device consists of 40 individual recording units (5 x 10 x 2 cm boxes), each featuring three infrared (IR) light emitting diodes (LEDs) positioned face to face with their respective receivers to detect insect movement. This device was used to record bug locomotory activity in all assays reported in this study. During the experiments, room temperature was kept at 27 + 1°C and illumination provided at 12:12 LL/DD (light intensity = 60 LUX). Fifth instar nymphs starved for 30 days after ecdysis were used in all experiments to grant an adequate bug motivation for the expression of spontaneous host search activity [START_REF] Ferreira | Activity and shelter-related behaviour in Rhodnius prolixus : the role of host odours[END_REF]. In each assay, nymphs were individually placed in the recording units that contained a piece of filter paper as a substrate lining the floor of each box (5 x 10 cm). Once an assay was set, the number of movement events was continually recorded for each individual.

A preliminary experiment in which 32 nymphs were released and their movements recorded for 72 hours, allowed comparing the number of movement events recorded for each of the three daily intervals. The corresponding dataset showed that the number of movements over the recorded interval showed less variability after the first day (data shown in Supporting Information Figure S1). As a consequence we chose to restrict subsequent data sampling to the second day.

Two experiments were performed to analyze whether different locomotory profiles could co-exist in our bug colony. A first scrutiny characterized the hourly activity profile of 548 nymphs through a 24h interval. In a second experiment, we selected a subsample of 65 nymphs reflecting the activity level diversity of the previous bug sample and sorted them by sex [START_REF] Espínola | Nota sobre diferenças sexuais em formas imaturas de Triatominae (Hemiptera, Reduviidae)[END_REF]. Afterwards, these insects were used to quantify the relative expression of the Rpfor gene by qPCR (RTq-PCR). In total, 32 males and 33 females were used for this second experiment.

RNA extraction and cDNA synthesis

We initially evaluated the expression of the Rpfor gene in samples made of pooled brains or fat bodies obtained from insects with different physiological status. Gene expression patterns reflected physiological changes similarly for both tissues (Supplementary Figure S2). Based on this, subsequent qPCR experiments focused on fat bodies, because samples from single individuals provided enough RNA for qPCR experiments. Fat body samples were dissected between 14-18h after the end of locomotory activity assays. Total RNA was extracted using TRIzol™ (Invitrogen, Carlsbad, California, USA) according to manufacturer's instructions. The total RNA obtained was treated with Turbo DNA-free kit (Invitrogen, Vilnius, Lithuania) to eliminate genomic DNA. Sample concentration was determined using a Qubit 2.0

Fluorometer (Life Technologies, Eugene, Oregon, USA). cDNA was synthesized from 500ng of mRNA using the M-MLV Reverse Transcriptase enzyme (Promega, Fitchburg, WI, USA) and a modified oligo dT primer (MgdT 5'-CGGGCAGTGAGCAACG (T12)-3') [START_REF] Ursic-Bedoya | Identification and characterization of two novel lysozymes from Rhodnius prolixus , a vector of Chagas disease[END_REF] under the following conditions: 37°C for 60min and 95°C for 5min.

Quantification of Rpfor gene expression

Specific primers for Rpfor (Supplementary Table S1) were used in qPCR reactions to evaluate gene expression levels in individuals showing different locomotory profiles.

All reactions contained 1µl of cDNA, 0.5µl of each primer (10µM), and 5µl of Platinum [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[END_REF]. Relative levels of expression for each insect were compared using α-tubulin (TUB) as a reference gene [START_REF] Paim | Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodnius prolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR[END_REF] (Supporting Information Table S1).

Statistical analysis

Hierarchical clustering and multivariate analyses

Two data matrices were built based on the activity profiles of individual nymphs. The first dataset encompassed the recordings obtained with 548 individuals throughout the 24h interval. The second dataset included the number of movement events and molecular data obtained with the sex-sorted nymphs. Data analyses were performed using the R-software (v3.4) on the Rstudio suite version 1.1.423 [START_REF] Team | RStudio: Integrated Development Environment for R[END_REF]. A hierarchical clustering dendrogram and its corresponding heatmap were generated using the heatmap3 package (v1.1.1). As we considered the absence of movement (joint absences) to be biologically informative, we opted to follow the workflow suggested by [START_REF] Anderson | Navigating the multiple meanings of β diversity: a roadmap for the practicing ecologist[END_REF]. This implied estimating the pairwise dissimilarities between the objects in the matrix based on non-transformed raw counts and Euclidean indices. For this purpose, the 'vegdist' function from the R-package 'vegan' (v2.5.2) was called within the heatmap3 function script.

To address the effect of potentially relevant biological variables accountable for the variance within the matrices, multivariate ordination tests of the RDA type (redundancy analysis based on an ordinary unweighted linear regression, and unweighted singular variable decompositions) were executed considering their respective explanatory matrices (dummy matrices). Briefly, for each of the evaluated datasets, the dummy matrices contained explanatory data such as sex [male/female] (for 65 nymphs); total locomotion counts during the 24h span; and normalized gene expression coefficients (for 65 nymphs). The analyses and corresponding biplots (scaling=2, correlation plots focusing on response variables) were generated using the 'rda' function from the 'vegan' package (v2.4.6). Finally, to assess whether the hypothesized constraints could significantly explain the variance within the datasets, and thus how the response and explanatory variables interact, permutation-based MANOVA tests (PERMANOVA) were performed using the 'adonis2' function within the 'vegan' package (same version aforementioned) executing 9,999 permutations [START_REF] Anderson | A new method for nonparametric multivariate analysis of variance[END_REF][START_REF] Anderson | PERMANOVA, ANOSIM, and the Mantel test in the face of heterogeneous dispersions: what null hypothesis are you testing?[END_REF]. This multivariate analysis also generates an effect-size estimate (R 2 ) of the variance proportion explained by each of the hypothesized explanatory variables. A similar hypothesis testing approach was executed by [START_REF] Liu | Statistical analysis of zebrafish locomotor response[END_REF], when addressing how multiple biological effectors might contribute differently to the activity measured for zebrafish larvae.

Rpfor gene expression: its correlation with sex and locomotor activity

Data depicting expression levels for the Rpfor gene in male and female nymphs were tested for normality using the Shapiro-Wilk test. As expression levels did not adjust to a normal distribution, results were compared by means of the Mann Whitney test. A GLM-ANOVA was performed to test whether Rpfor expression and sex would influence the cumulative locomotory activity (24h). Then, the Spearman's correlation coefficient was used to examine whether the cumulative activity was correlated to the relative expression of Rpfor gene for each sex separately.

RESULTS

The median number of locomotion events recorded for the 548 nymphs during the 24h interval was of 246.5 (0-2,753). Figure 1 presents the activity profile obtained

showing the characteristic two peaks described by previous reports studying triatomine bugs [START_REF] Guarneri | The effect of temperature on the behaviour and development of Triatoma brasiliensis[END_REF][START_REF] Lazzari | Circadian organization of locomotion activity in the haematophagous bug Triatoma infestans[END_REF][START_REF] Lorenzo | Activity pattern in relation to refuge exploitation and feeding in Triatoma infestans (Hemiptera: Reduviidae)[END_REF][START_REF] Marliére | Trypanosomes modify the behavior of their insect hosts: effects on locomotion and on the expression of a related gene[END_REF][START_REF] Pavan | Rhodnius prolixus and R. robustus (Hemiptera: Reduviidae) nymphs show different locomotor patterns on an automated recording system[END_REF]. These peak hours (8 and 20h) concentrated approximately 18 and 16% of the movement events, respectively. In general, a higher activity was observed during the first four hours of the scotophase, which represented approximately half of all locomotory activity expressed. However, as indicated by the amplitude between min and max values, the number of movement events recorded varied expressively (Figure 1). showing an average of 251.2 (82-796) and 59.5 events (0-299), respectively. Of the total set of individuals, 30.7% belonged to block 1, 10.2% to block 2 and 59.1% to block 3 (subgroup A 27.2 and B 31.9%). The most active bugs were assigned to block 2, while the least active ones grouped in block 3B, which included those that remained inactive through the assay.

The RDA analysis using cumulative individual activity (i.e., the total number of locomotion events per individual over 24h, henceforth referred to as total activity) as constraining variable, accounted for 42.59% of the total variance within the axes shown in Figure 3 (RDA1; and PC1-summarizing unconstrained variance) . From that, 29.27% of the variation was due to the proposed explanatory variable (total activity), and its influence upon the objects and their correlations is represented along axis RDA1. The length of the vector is a measure of the magnitude of the response variable (total activity). The dispersion observed between individuals points to the existence of different activity profiles. Insects with low or null activity were positioned at the base of the total activity vector. Thirty-seven nymphs (6.7%) that presented zero locomotion counts for the whole 24h interval evaluated clustered at an ≈180° angle regarding the total activity vector (scores converging approximately around the [-0.4,4.8e-2] coordinates). As the number of movement events increased, bugs were distributed in the two dimensions following a bimodal pattern in which individuals with similar total activity counts (but different temporal profiles) separated along the ordinate space (examples of individual per -hour activity profiles are shown in Supplementary Figure S3). The further they followed the explanatory vector, the higher the magnitude of their response to this constraint, and their dissimilarity with the other individuals. The hourly intervals showing a stronger effect on total locomotion were 20, 21, 22, 23, and 24 (grouped below the activity vector). The hourly intervals corresponding to the photophase (from 9 to 19) grouped in the upper right quadrant of the ordinate space. The hourly intervals preceding the beginning of the photophase grouped close to the activity vector indicating that they share similar characteristics. In fact, this interval (except for 8h) presented low and uniform locomotory activity for all nymphs.

Searching for factors potentially related to the locomotory profiles, a new RDA was performed based on the hourly activity of 65 R. prolixus nymphs over 24h, and considering their total activity, sex and Rpfor gene expression as explanatory variables (Figure 4). According to the RDA, an estimated 45.32% of the variance among the profiles could be explained by the three variables. Total activity was the variable with the largest vector magnitude (Figure 4). The position of the vector representing Rpfor expression (≈-270°) suggests a negative relation with the activity vector (Figure 4). Males and females spread out differently along the activity vector, with the former dispersing preferentially through the space above the activity vector and the latter through the lower space. The position of the geometric means (centroids) in relation to the activity vector suggests different profiles of locomotory activity for males and females. We tested this hypothesis of sexually different locomotory profiles in a third dataset (including the hourly locomotion profiles of 175 individuals shown as Supplementary Figure S4) and obtained a similar outcome. We therefore compared the levels of Rpfor expression in males and females and found that Rpfor gene expression was significantly higher for female nymphs (Supplementary Figure S5, Mann Whitney, p=0.019).

A PERMANOVA test was then performed to address the significance with which each of the proposed variables explained the response matrix variations (Table 1).

Globally, each of the variables tested, and the interaction between them drove 66.29% of the variance encompassed in the response matrix. Total locomotory activity was the most significant explanatory variable affecting locomotion profiles, presenting the largest effect size (Table 1; PERMANOVA, p=0.001). Also significant, but with smaller effect sizes, were sex, Rpfor expression and the interaction between sex and total activity (Table 1; PERMANOVA, p=0.004, p=0.005 and p=0.01, respectively). The significant interaction between sex and total activity indicated that the effects of total activity on the locomotion profiles were influenced by nymph sex.

As bug cumulative activity was the main factor influencing the different locomotion profiles, we tested whether the levels of activity were affected by gene expression and sex. We found a significant effect of sex and Rpfor expression on total bug activity (Table 2; GLM-ANOVA, p<0.0001 for both parameters). The significant interaction between sex and Rpfor expression (Table 2; GLM-ANOVA, p<0.0001) indicated that the effects of Rpfor expression on the levels of total activity were highly affected by nymph sex. We further tested whether total activity and gene expression were correlated in each of the sexes. A positive, but non-significant, correlation between total activity and Rpfor expression was found for female nymphs (Spearman correlation, rS=+0.32; p=0.067). Interestingly, a significant negative correlation between total male activity and Rpfor expression was detected (Spearman correlation, rS=-0.67; p>0.0001).

DISCUSSION

As reported in the literature [START_REF] Guarneri | The effect of temperature on the behaviour and development of Triatoma brasiliensis[END_REF][START_REF] Lazzari | Circadian organization of locomotion activity in the haematophagous bug Triatoma infestans[END_REF][START_REF] Lorenzo | Activity pattern in relation to refuge exploitation and feeding in Triatoma infestans (Hemiptera: Reduviidae)[END_REF][START_REF] Marliére | Trypanosomes modify the behavior of their insect hosts: effects on locomotion and on the expression of a related gene[END_REF], the insects tested in this study showed two peaks of activity in both, the beginning of the scotophase and photophase. As expected, half of all locomotion events were concentrated in the first hours of the scotophase. Nevertheless, the pairwise comparison of hourly movement events uncovered clearly distinct activity profiles. The hierarchical clustering divided our locomotion dataset into three principal branches as a result of the activity levels recorded and the hour in which movement was detected. Interestingly, more active insects showed the highest dissimilarities, while insects that tended to stay motionless showed higher homogeneity. Our results suggest that different locomotory strategies co-exist in R. prolixus . We acknowledge the intrinsic limitations of laboratory colonies in terms of genetic variability. Therefore, it would be relevant to test whether the locomotion profiles of sylvatic R. prolixus resemble those reported here.

As expected, the multivariate analyses (HC and RDA) suggested that the temporal dimension was the main factor influencing activity levels. Interestingly, two unknown features of kissing-bug locomotory patterns were revealed by applying these analyses. Firstly, we determined that our robust bug sample consists of individuals showing differentiated levels of activity, as shown in the three main branches created by the dendrogram. At the extremes, approximately 7% of the nymphs did not move, while 3% of them produced more than a thousand movement events during the same interval. The very different activity profiles reported in the dendogram may represent coexisting energy investment strategies and their ecological relevance deserves to be studied. Nutritional shortage scenarios are common in the sylvatic ecotopes of triatomines [START_REF] Noireau | Flight and nutritional status of sylvatic Triatoma sordida and Triatoma guasayana[END_REF][START_REF] Sarquis | Ecology of Triatoma brasiliensis in northeastern Brazil: seasonal distribution, feeding resources, and Trypanosoma cruzi infection in a sylvatic population[END_REF]. Under such circumstances, low activity could be advantageous to avoid consuming nutritional reserves while waiting for a blood-source to arrive to the ecotope. As an alternative strategy, intense locomotion could allow other individuals to approach a distant host, overcoming their nutritional shortage at increased predation risks induced by exposure outside shelters.

The second uncovered feature was the presence of individuals with similar activity levels that grouped separately according to dissimilarities in their temporal profiles. More specifically, their main activity bouts were expressed at different times.

In order to explore the dataset in more detail we decided to focus on the expression of activity during the photophase. To avoid considering activity belonging to the tails of the main peaks, we excluded the two hours after lights on and before lights off from this calculation. This revealed that 21.9% of the insects were active in half of the hours belonging to this interval. Furthermore, 1.8% of them moved in all hours of the photophase. This temporal locomotory spread may also be an advantageous strategy in a nutritional shortage scenario, as foraging outside conventional hours would allow feeding on nocturnal vertebrates when these enter their resting state. In fact, opossums are fundamentally nocturnal and their blood is consistently detected in the gut of sylvatic insects [START_REF] Hernández | Untangling the transmission dynamics of primary and secondary vectors of Trypanosoma cruzi in Colombia: parasite infection, feeding sources and discrete typing units[END_REF][START_REF] Sasaki | Host feeding profiles of Rhodnius prolixus and Triatoma dimidiata in Guatemala (Hemiptera: Reduviidae: Triatominae)[END_REF]. It is important to acknowledge that this was not the predominant locomotion profile, suggesting that being active during daytime increases predation risk. It is also worth pointing out that this experiment was performed with individuals that lacked access to shelters and co-specific aggregations. According to our current knowledge about shelter use by kissing-bugs, most individuals tend to group inside a refuge each night and only a small fraction of them leaves the protected places spontaneously [START_REF] Ferreira | Activity and shelter-related behaviour in Rhodnius prolixus : the role of host odours[END_REF][START_REF] Lorenzo | Activity pattern in relation to refuge exploitation and feeding in Triatoma infestans (Hemiptera: Reduviidae)[END_REF]. In this sense, future studies are necessary to address whether insects that leave shelters in the absence of host cues belong to any of the different activity profiles seen here. To confirm the existence of individuals which are active during the day, one should also evaluate the dynamics of the use of shelters during the light phase, in both the presence and absence of host cues.

Our results are also suggestive of sex-specific behavioral profiles expressed by R.

prolixus 5 th instar nymphs. Sexually dimorphic locomotion patterns have been reported previously for house flies and Drosophila melanogaster [START_REF] Bahrndorff | The effects of sex-ratio and density on locomotor activity in the house fly, Musca domestica[END_REF][START_REF] Belgacem | Neuroendocrine control of a sexually dimorphic behavior by a few neurons of the pars intercerebralis in Drosophila[END_REF][START_REF] Buchan | Effect of temperature and different sex ratios on physical activity and life span in the adult housefly, Musca domestica[END_REF][START_REF] Helfrich-Förster | Differential control of morning and evening components in the activity rhythm of Drosophila melanogaster -sex-specific differences suggest a different quality of activity[END_REF][START_REF] Martin | Temporal pattern of locomotor activity in Drosophila melanogaster[END_REF][START_REF] Ragland | Mating behavior, physical activity and aging in the housefly, Musca domestica[END_REF]. This is also the case for triatomines, for which adult bug behavior differs according to sex, as male kissing-bugs show active locomotory responses to sexual signals, while females do not [START_REF] Manrique | The sexual behaviour of Chagas' disease vectors: chemical signals mediating communication between male and female Triatomine bugs[END_REF]. Indeed, the motivation to leave shelters is strikingly different for male and female kissing-bugs [START_REF] Pontes | Female odours promote the activation of sheltered kissing bug Rhodnius prolixus males and modulate their orientation[END_REF]. These authors showed that a relevant proportion of adult female bugs leaves shelters spontaneously and forages in the absence of host cues. Notably, the presence of males in the same environment does not alter this profile [START_REF] Pontes | Female odours promote the activation of sheltered kissing bug Rhodnius prolixus males and modulate their orientation[END_REF]. On the other hand, female odors promote the activation and orientation of adult males that otherwise do not seem to leave shelters. Ours is the first study reporting sex-specific locomotory activity profiles in immature triatomines. As mentioned, bugs were not exposed to shelter-associated stimuli or co-specifics, which would induce akinesis and aggregation behavior.

Further studies should clarify whether sex-specific differences in locomotory activity expressed by nymphs relate to the search for sexual signals by immature male insects.

The expression of Rpfor influenced spontaneous locomotory activity significantly, reinforcing its suggested role in the modulation of foraging profiles. In addition, we found that the Rpfor gene affects total bug activity in a sex-dependent way. In fact, the inverse correlation between the total activity vector of males and Rpfor expression, suggests that decreased Rpfor expression induces increased male locomotory activity. Positive [START_REF] Ben-Shahar | Influence of gene action across different time scales on behavior[END_REF][START_REF] Lucas | The locust foraging gene[END_REF][START_REF] Pereira | Mutations in the larval foraging gene affect adult locomotory behavior after feeding in Drosophila melanogaster[END_REF][START_REF] Tobback | Ageand taskdependent foraging gene expression in the bumblebee Bombus terrestris[END_REF] and inverse [START_REF] Ingram | Taskspecific expression of the foraging gene in harvester ants[END_REF], George et al., 2018;[START_REF] Tobback | Cloning and expression of PKG, a candidate foraging regulating gene in Vespula vulgaris[END_REF] correlations between foraging expression and locomotory activity have been reported.

In conclusion, cluster analysis evinced distinguishable locomotion patterns that may represent diverse energy investment strategies .

Then, multivariate analyses have provided original perspectives on the molecular control of the expression of kissing-bug locomotory activity. Furthermore, they allowed us to identify unexpected sex-specific patterns of 5 th instar nymph locomotion, which are apparently affected by the differential expression of the Rpfor gene.

Competing interests

No competing interests declared.

  SYBR green qPCR SuperMix-UDG (Invitrogen, Carlsbad, California, USA) in a final volume of 10µl. Experiments were conducted at the qReal-Time PCR Facility -RPT09D PDTIS/René Rachou Institute/FIOCRUZ MG in a ViiA 7 System (Applied Biosystems, Foster City, California, USA) under the following conditions: 10min at 96°C, followed by 40 cycles of 15sec at 95°C and 20sec at 60°C. Melting curve analyses confirmed the specificity of the reaction. All individual cDNA samples were evaluated in two technical replicates. qPCR results were analyzed by calculating the 2 -ΔΔCt

  To search for patterns based on individual locomotory activity profiles, we applied a multivariate analysis to generate a pairwise Euclidean distance matrix among hourly locomotion counts during the 24h interval. A hierarchical clustering test was executed based on the recordings of individual activity (i.e., number of locomotion events per individual per hour). As seen in Figure2, three major blocks encompassing individuals with resembling dissimilarity patterns were identified based on dendrogram topology, heatmap intensity, and activity scatter plot (referred to as blocks 1, 2 and 3). Block number 1 (blue) contained 168 insects presenting an average of 461 movement events (113-1,468), while the 56 individuals from block 2 (green) showed an average of 918 events (410-2,753), and those in block 3 (pink, including 324 insects) an average of 147 events (0-796). Furthermore, by subdividing block 3 along its first node this cluster split in groups A (149) and B (175 individuals),

Figure 1 .

 1 Figure 1. Hourly median of movement events shown by 5 th instar Rhodnius prolixus nymphs over a 24h interval. The white and gray bars represent the photophase and scotophase, respectively. Data are presented as the median, 25%-75% quartiles, minimal and maximal values of the number of movement events per hour based on data collected with 548 insects.

Figure 2 .

 2 Figure 2. Hierarchical clustering of triatomine locomotory profiles based upon number of movements per hour and their pairwise Euclidean distances. The heatmap depicts the degree of dissimilarity amongst the 548 individuals during 24h. The middle panel, between the heatmap and the dendrogram, presents a scatter plot of the total activity recorded for each individual throughout the selected interval. The colored areas and trend line in the scatter plot aid visualizing the dendrogram topology as they discriminate deeper node tips from each other according to the proposed blocks based on total locomotion counts and dissimilarity patterns. Block number 1 (blue) contains 168 insects, block 2 (green) is composed by 56 individuals, and block 3 (pink) by 324 insects. Block 3 was subdivided along its first node in groups A and B, with 149 and 175 individuals, respectively).

Figure 3 .

 3 Figure 3. Redundancy analysis (RDA) biplot considering total activity throughout a 24h interval as explanatory variable. The RDA depicts how individuals dispersed on the (constrained) ordination space based upon Euclidean dissimilarities between their locomotion profiles. The patterns of variation within the dataset of response variable measures were constrained by the total activity counts recorded during a 24h interval (vector pointing right along the horizontal axis). In total, 42.59% of the variance is depicted in the biplot. From it, the proposed explanatory variable (total

  Insects were maintained by the Vector Behavior and Pathogen Interaction Group at René Rachou Institute, Belo Horizonte,

	Brazil. Experimental bugs were fed citrated rabbit blood obtained from CECAL
	(Fiocruz, Rio de Janeiro, Brazil) offered through an artificial feeder at 37°C,
	alternating with blood from anesthetized chicken. The latter were anesthetized with
	intraperitoneal injections of a mixture of ketamine (20 mg/kg; Cristália, Brazil) and
	detomidine (0.3 mg/kg; Syntec, Brazil), under license number LW-61/2012
	(Committee for Ethics in the Use of Animals, CEUA-FIOCRUZ).

Table 2 .

 2 GLM analysis of the effects of Rpfor expression and sex on the cumulative activity of Rhodnius prolixus nymphs .

	Factor	DF	R 2	F	Pr(>F)
	Total_activity	1	0.5455	93.842	0.001	**
	Sex	1	0.0294	5.059	0.004	**
	Rpfor	1	0.0424	7.305	0.005	**
	Sex: Rpfor	1	0.0083	1.439	0.200
	Sex:Total_activity	1	0.0287	4.948	0.011	*
	Rpfor :Total_activity	1	0.0083	1.427	0.215
	Residual	58	0.3371
	Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
	Evaluated formula = matrix65v24 ~(((Sex * FOR)) + (Sex * Total_activity) + (FOR *
	Total_activity)), data = dummy65iexp, permutations = 999, method = "euclid", by =
	"terms").
		Estimate Std. Error Z Value	Pr(>z)
	(Intercept)	5.4849	0.0217	252.60	<2e-16 ***
	Sex	1.8700	0.0279	66.95	<2e-16 ***
	Rpfor	1.4693	0.0676	21.73	<2e-16 ***
	Sex: Rpfor	-7.3204	0.1237	-59.16	<2e-16 ***
	Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.

Acknowledgements

AAG and MGL were supported by CNPq productivity grants. MGL received funding from Le Studium (Short Term Contract of Employment N°2017-2001-179 -Y17F16). The authors thank the Program for Technological Development in Tools for Health-PDTIS-FIOCRUZ for use of its facilities.

Funding

This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, grant numbers CRA-APQ-00569-15 and CRA-PPM-00162-17), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number 441999/2014-0 ), Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCTEM/CNPq, grant number 465678/2014-9), Programa Estratégico de Apoio a Pesquisa em Saúde (PAPES VI/FIOCRUZ, grant number 407614/2012-5).

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001.

Locomotion profile dissimilarities were constrained by explanatory variables accounting for total activity during 24h, sex, and Rpfor gene expression profiles. In total, 45.32% of the variance within the matrix is driven by the proposed variables.

From it, 98.09% of the constrained variance resolved along the horizontal axis (RDA1), whereas 1.52% is resolved along RDA2. Male and female locomotion profiles were found to differ significantly based on the permutational MANOVA.