Saoussen Asma Kerbiche
email: asma.kerbiche@gmail.comsaoussen.bj@laposte.netezzeddine.zagrouba@fsm.rnu.tn

Ben Jabra

Ezzeddine Zagrouba

Vincent Charvillat
email: vincent.charvillat@enseeiht.fr

Video Watermarking Based on Interactive Detection of Feature Regions

Keywords: watermarking, crowdsourcing, robustness, invisibility. I

Video watermarking is very important in many areas of activity and especially in multimedia applications. Therefore, security of video stream has recently become a major concern and has attracted more and more attention in both the research and industrial domains. In this perspective, several video watermarking approaches are proposed but, based on our knowledge, there is no method which verified the compromise between invisibility and robustness against all usual attacks. In our previous work, we proposed a new video watermarking approach based on feature region generated from mosaic frame and multi-frequential embedding. This approach allowed obtaining a good invisibility and robustness against the maximum of usual attacks. In our future work, we propose to optimize the choice of the region of interest by using crowdsourcing technique. This last one is an emerging field of knowledge management that involves analyzing the behavior of users when they view a video to automatically deduct the regions of interest.

INTRODUCTION

The fast development of the Internet in recent years has eased the process of coping, transmitting, and distributing digital data such as image, 3D meshes and video. In fact, the recent decade has seen the emergence of video-based application technologies, such as wireless video, video conferencing, and videophones. As with other types of multimedia data, it was necessary to find new techniques for visualization, compression, indexing, and also the security of this type of media to enable the protection of rights, authentication and data integrity during transfer on a given communication channel. Therefore, the need for protection from piracy and illegal use rises more and more. The best technique to protect digital video from this manipulation is watermarking. It consists of embedding a signature into data and to try to detect it after any manipulation done on marked data. Usually, signature must be imperceptible and should resist to malicious attacks which try to destroy or to remove it. Several studies have been made to develop robust and invisible video watermarking methods [START_REF] Paul | Review of Robust Video Watermarking Techniques[END_REF]. These methods differ mainly in the insertion area [START_REF] Rajab | Video Watermarking Algorithms Using the SVD Transform[END_REF] (spatial or frequency) and the type of the treated stream [START_REF] Campisi | Video watermarking in the 3D-DWT domain using perceptual masking[END_REF] (compressed or uncompressed). To improve the invisibility and the robustness of the watermark, some methods are based on the choice of region of interest where the embedding will be done. This choice can be based on many tests applied on different feature regions [START_REF] Kerbiche | A robust video watermarking based on image mosacing and multi-frequential embedding[END_REF] or using a specific technique [START_REF] Koubaa | Collusion, MPEG4 compression and frame dropping resistant video watermarking[END_REF]. In our work, we propose to use crowdsourcing technique that involves analyzing the behavior of users when they view a video to automatically deduct the regions of interest. The remainder of this paper is organized as follows: in Section 2, video watermarking and crowdsourcing technique are presented while Section 3 describes the proposed watermarking method based on interactive detection of regions of interest using crowdsourcing technique. Finally, conclusion is drawn in Section 4.

II. RELATED WORK

A. Watermarking

Several techniques of watermarking video have been proposed in the literature. We chose to classify them according to two criteria. The first one is the original video format which can be compressed or uncompressed. In fact, watermarking can be applied to compressed stream where the insertion is done during the compression process or after compression [START_REF] Mohaghegh | H.264 copyright protection with motion vector watermarking[END_REF], [START_REF] Bas | Image watermarking; An evolution to content based approaches[END_REF], [START_REF] Joumaa | Performance of an application video watermarking scheme using informed techniques[END_REF]. For the uncompressed video, four classes of embedding methods can be applied (some of them can also be used on compressed video). The first class is derived from frame by frame watermarking that consider video as a succession of images and consists of applying still images watermarking algorithms [START_REF] Chae | Data hiding in video[END_REF], [START_REF] Merchant | Watermarking of video data using integer-to-integer discrete wavelet transform[END_REF]. The second one is the spatio-temporal schemes where video is defined as a 3D signal considering the temporal dimension in video sequences. These schemes decompose the video by performing spatial 2D transform on individual frames followed by 1D transform in the temporal domain [START_REF] Deguillaume | Robust 3D DFT video watermarking[END_REF], [START_REF] Lim | Digital video watermarking using 3D-DCT and intracubic correlation[END_REF], [START_REF] Kim | A new digital video watermarking using the dual watermark images and 3D DWT[END_REF]. The third class is the temporal schemes that insert the signature in the temporal domain by modifying only the low spatial frequencies [START_REF] Vinod | Motion-compensated inter-frame collusion attack on video watermarking and a countermeasure[END_REF], [START_REF] Vinod | Assessing motion-coherency in video watermarking[END_REF], [START_REF] Su | Statistical invisibility for collusion resistant digital video watermarking[END_REF]. Finally, the last class is based on mosaic frame generated from the original video [START_REF] Doerr | Secure background watermarking based on video mosaicing[END_REF], [START_REF] Koubaa | Collusion, MPEG4 compression and frame dropping resistant video watermarking[END_REF]. This last one selects an interesting area where the mark should be embedded. In fact, mosaïcing allows the insertion of the same mark into the same pixels which represent the same physical point.

++

The second criterion is embedding domain where signature can be inserted directly on video by modifying its pixels [START_REF] Seddik | A New Spatial Watermarking Method, based on a Logarithmic transformation of An Encrypted embedded Mark[END_REF] or by modifying some video transformations like DCT (Discrete Cosine Transform), DWT (Discrete Wavelet Transform) and SVD transform (Singular Value Decomposition) [START_REF] Liu | A robust video watermarking scheme based on DCT[END_REF], [START_REF] Chan | A DWT-based Digital Video Watermarking Scheme with Error Correcting Code[END_REF]. The scheme disperses the watermark in the spatial domain of the video frame, hence making it very difficult to remove the embedded watermark. In our previous work, after a comparative study of these classes [START_REF] Kerbiche | A robust video watermarking based on image mosacing and multi-frequential embedding[END_REF], we proposed a new watermarking schema based on mosaic and multi-frequentiel embedding algorithm using wavelet, DCT and SVD transforms. In fact, it consists to generate mosaic from original video. Then, the region where the objects move is selected to be marked. Finally, signature is inserted using wavelet, DCT and SVD transforms. This choice allows obtaining robustness against various types of attacks, such as geometric transformations (rotation, zooming), cropping vertices, MPEG4 part X (h.264 Advanced Video Coding (AVC)) compression, noise, frame suppression and collusion.

B. Crowdsourcing

With the sweeping progress of Web 2.0 technologies and capabilities, many socio-technical systems have attracted attention from both practitioners and scholars. Crowdsourcing is a new emerging Web 2.0 based phenomenon and becomes a recognized sourcing mechanism for problem solving in organizations and societies by outsourcing problems to an undefined entity or the 'crowd'. For that, crowdsourcing research has become a dynamic and vibrant research area, and has been steadily growing over the years.

The term crowdsourcing was first coined by Howe, in a Wired Magazine article in June 2006 [START_REF] Howe | The rise of crowdsourcing[END_REF]: "Simply defined, crowdsourcing represents the act of a company or institution taking a function once performed by employees and outsourcing it to an undefined (and generally large) network of people in the form of an open call. This can take the form of peer-production (when the job is performed collaboratively), but is also often undertaken by sole individuals. The crucial prerequisite is the use of the open call format and the large network of potential laborers".

In essence, crowdsourcing is based on a simple, but powerful, concept: virtually everyone has a potential to plug in valuable information [START_REF] Greengard | Following the Crowd[END_REF]. It seeks to mobilize competence and expertise, which are distributed among the crowd and has different forms [START_REF] Zhao | Evaluation on crowdsourcing research: Current status and future direction[END_REF].

Crowdsourcing is not exclusive for business purposes. In fact, many non-profit organizations have adopted it as an effective model for problem-solving [START_REF] Brabham | Crowdsourcing as a model for problem solving: an introduction and cases[END_REF], [START_REF] Brabham | Moving the crowd at threadless: motivations for participation in a crowdsourcing application[END_REF]. In addition to having gained great attention and interest from the industry, crowdsourcing has also gained attention from the academic community.

Indeed, several recent studies are based on crowdsourcing technique. Xie et al. [START_REF] Xie | Learning User Interest for Image Browsing on Small-formfactor Devices[END_REF] propose new method to detect user interest maps and extract user attention objects from the image browsing log using crowdsourcing where ten subjects were selected to take part in this study, and the criterion for their selection was that they should be very familiar with the use of computers and cell phones before the study. A smart image viewer was then developed based on user interest analysis and a second experiment was carried out to study how users behave with such a viewer. This approach is more efficient than image-analysis based methods and can better represent users' actual interest. Based on the fact that the viewing experience on the mobile devices can be improved by determining important and interesting regions within the video (regions of interest, ROIs) and displaying only the ROIs to the viewer, Carlier et al [START_REF] Carlier | Crowd-sourced Automatic Zoom and Scroll for Video Retargeting[END_REF] propose an alternative paradigm to infer ROIs from a video by crowdsourcing from a large number of users through their implicit viewing behavior using a zoom and pan interface, and infer the ROIs from their collective wisdom. A retargeted video, consisting of relevant shots determined from historical users' behavior, can be automatically generated and replayed to subsequent users who would prefer a less interactive viewing experience. A user study with 48 participants shows that this automatically retargeted video is of comparable quality to one handcrafted by an expert user.

III. PROPOSED METHOD

The study of watermarking and crowdsourcing recent works shows that these two areas can be combined to propose a new watermarking approach which presents a high level of robustness against the most important attacks. In fact, our work aims to develop new robust approaches to introduce signatures in videos. Our idea consists in a first step to understand the visual content of the original video and then to select feature regions to embed signature. To achieve our goal, crowdsourcing technique will be used. Although, the concept of crowdsourcing is based on sharing media to the public, this can cause confidentiality problems and can damage watermarking process. To avoid this problem, we thought to generate a video summary and to share it to a fixed number of selected users.

The proposed approach is decomposed to three main stages: video summarization, interactive detection of feature regions using crowdsourcing technique and signature insertion. General architecture of the proposed approach is presented by Figure 2.

A. 1st Step: Video Summarization

To avoid confidentiality problems that can be caused by sharing the original video, its summary will be generated using the approach proposed in [29]. This last one is an object-based technique which allows extracting a relatively small number of still key-frames in order to summarize the salient visual content of a video. This method is based on spatial segmentation of each frame in order to detect the important events. Indeed, the extraction of key-frames will be facing a much more semantic criterion so that each extracted key-frame present an important event such as the appearance and/or the disappearance of significant objects.

B. 2nd Step: Feature Regions Detection

After video summarization, feature regions must be selected to embed signature. To detect these regions, crowdsourcing technique is chosen. In fact, this technique is an emerging field of knowledge management which allows analyzing the behavior of users when they watch a video to automatically deduct the regions of interest. Indeed, the combination of the analysis of visual content and interactive use of the identified data can improve the detection of interest visual objects in a video. The method proposed in [START_REF] Campisi | Video watermarking in the 3D-DWT domain using perceptual masking[END_REF] and explained in the previous section, has been used for this step. To ensure the confidentiality of our video and to avoid video hacking, we choose to interact with an X number of selected users in our laboratory.

C. 3rd Step: Watermarking

After extracting user attention objects, the signature will be inserted in the N feature regions selected from second step. To embed signature, our multi-frequential watermarking scheme [START_REF] Kerbiche | A robust video watermarking based on image mosacing and multi-frequential embedding[END_REF] based on DWT, DCT and SVD transforms will be used. In fact, the regions selected by users are the most important regions in the video and their destruction will cause the destruction of the whole video. Thereby, the insertion in these selected regions will ensure a greatest robustness of our watermark. General architecture of the embedding step is presented by Figure 2.

D. Test

The reliability of the developed method will be tested by evaluating the two main criterions: robustness and invisibility. In fact, for the robustness we will test the resistance of the watermark against compression phases, collusion, Cropping and several main types of video attacks that could destroy the watermark. For invisibility criterion and in order to enhance our tests, crowdsourcing process will be used. user will be carried.

IV. CONCLUSION AND FUTUR WORK

Crowdsourcing has become a dynamic and vibrant research area, and has been steadily growing over the years. In fact, it can be applied to a wide variety of problems and ever more classes of applications. For this reason, we proposed in this paper a new video watermarking based on crowdsourcing technique to select the most interesting feature regions. These last ones will be used to insert signature. In fact, interactive detection of feature regions using Crowdsourcing technique will guaranty a high level of robustness and invisibility of marked video. The robustness of the proposed method will be verified after application of various types of attacks such as geometric transformations (rotation, zooming), cropping vertices, MPEG4 compression, noise, frame suppression and collusion. For invisibility, we will use three metrics (peak signal to noise ratio (PSNR), Haussdorff distances and correlation) and a user study will be done to measure marked video quality.

This method can be extended to 3D video and can be used by all watermarking applications such as video indexing.

Figure 1 .

 1 Figure 1. General architecture our proposed method.

Figure 2 .

 2 Figure 2. General architecture of the embedding step.

TABLE I .

 I ADVANTAGES AND INCONVENIENCES OF EACH CLASS ACCORDING TO VIDEO FORMAT

	Methods	Advantages	Inconveniences	Invisibility
		Minimum	May	be
		insertion time	ineffective
		and	Avoids	against	a
	Frame by frame watermarking	compression steps which can and decompression	MPEG-4 the inserted compression that will destroy	+
		degrade	the	signature.
		images of the	Possibility	of
		sequence.	artifacts
	Spatio-temporal schemes	Robustness changes against temporal	Unblinded capacity detection low insertion and	++
	Temporal Schemes	The mark value is constant for a given image, another. one image to but vary from	Fragile watermark changes. temporal against	+
				Fragile
	Compressed Video	If the encoding is complex detection is not.	watermark may disappear against motion estimation and	++
				at re-coding.
		Robust against
	Mosaic Schemes	MPEG-4 compression, collusion attacks and	Not against MPEG-robust 2 compression.
		deleting images

TABLE II

 II

	.	COMPARISON OF EMBEDDING DOMAIN CLASSES
		Methods	Advantages	Invisibility
			The most robust
		DCT	against		++
			compression
			The most robust
	Frequency Domain	DWT	against compression Robust against	++
			compression,
		SVD	rotation, noise	++
			and	deleting
			frames
	Spatial Domain	LSB Correlation-Based techniques	Not against collusion, robuste compression, noise...	+


CRVOI Muriel Dietrich
email: muriel.dietrich@gmail.com

David A Wilkinson

Aude Benlali

Erwan Lagadec

Beza Ramasindrazana

Koussay Dellagi

Pablo Tortosa

Leptospira and paramyxovirus infection dynamics in a bat maternity enlightens pathogen maintenance in wildlife

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

Introduction

Seasonal changes can have important effects on infectious disease dynamics [START_REF] Altizer | Seasonality and the dynamics of infectious diseases[END_REF]. Of the many driving factors of infection that demonstrate seasonal cycles, annual reproduction has been identified as having a significant impact on the dynamics of various host-parasite interactions, particularly in animal species that are gregarious during the reproductive season [START_REF] Altizer | Seasonality and the dynamics of infectious diseases[END_REF]. Indeed, during this period, aggregation within colony, increased metabolic activity and the highly synchronized birth of newborns (i.e. birth pulse) lead to increased numbers of susceptible hosts and hence boost the potential for transmission of infectious agents (e.g. [START_REF] Hosseini | Seasonality and wildlife disease: how seasonal birth, aggregation and variation in immunity affect the dynamics of Mycoplasma gallisepticum in house finches[END_REF]. Investigating the infection dynamics during host breeding seasons may therefore help to better identify major mechanisms of pathogen maintenance within wild animal populations.

Bats play important ecological roles in prey and predator cycles, arthropod suppression, seed dispersal, pollination, as well as nutrient distribution and recycling [START_REF] Kunz | Ecosystem services provided by bats[END_REF].

Additionally, bats have been identified as natural reservoirs of many microbial agents, including pathogens of threat to human health [START_REF] Wibbelt | Emerging diseases in Chiroptera : why bats ?[END_REF]. Their ability to enter torpor or hibernation, the large sizes of their gregarious groups and possibly some peculiarities in their immune system, have been suggested to facilitate maintenance of infections in these unique long-lived flying mammals [START_REF] Calisher | Bats: important reservoir hosts of emerging viruses[END_REF]. The role of seasonal breeding in bat infection dynamics has been the subject of several recent studies [START_REF] Amengual | Temporal dynamics of European bat lyssavirus type 1 and survival of Myotis myotis bats in natural colonies[END_REF][START_REF] Turmelle | Ecology of Rabies Virus Exposure in Colonies of Brazilian Free-Tailed Bats (Tadarida brasiliensis) at Natural and Man-Made Roosts in Texas[END_REF][START_REF] Drexler | Amplification of emerging viruses in a bat colony[END_REF][START_REF] Field | Hendra virus infection dynamics in Australian fruit bats[END_REF][START_REF] Mühldorfer | Diseases and causes of death in European bats: dynamics in disease susceptibility and infection rates[END_REF], mostly focusing on viruses. For example, lyssaviruses and henipaviruses have been observed to exhibit robust and specific amplification in maternity colonies [START_REF] Drexler | Amplification of emerging viruses in a bat colony[END_REF][START_REF] George | Host and viral ecology determine bat rabies seasonality and maintenance[END_REF]. Furthermore, although multi-pathogen interactions have been shown to drive infection dynamics in other host-parasite models (e.g. [START_REF] Telfer | Species interactions in a parasite community drive infection risk in a wildlife population[END_REF], very few studies have considered the dynamics of multiple infections in natural bat populations [START_REF] Drexler | Amplification of emerging viruses in a bat colony[END_REF][START_REF] Mühldorfer | Diseases and causes of death in European bats: dynamics in disease susceptibility and infection rates[END_REF].

Leptospirosis is considered as the most common bacterial zoonosis in the world and is caused by pathogenic spirochetes of the genus Leptospira [START_REF] Adler | Leptospira and leptospirosis[END_REF]. The incidence of human leptospirosis is high in tropical islands, particularly in the Indian Ocean Islands where some of the highest human incidences have been reported to date [START_REF] Pappas | The globalization of leptospirosis: worldwide incidence trends[END_REF]. Chronic carrier animals are characterized by persistent leptospire colonization of renal tubules, which are then shed for months or years in urine and contaminate the environment. Human infection occurs either directly from contact with animal reservoirs or indirectly from contaminated soil or water [START_REF] Adler | Leptospira and leptospirosis[END_REF]. Rodents are classically recognized as the most significant reservoirs for the maintenance and dissemination of leptospires worldwide [START_REF] Bharti | Leptospirosis : a zoonotic disease of global importance[END_REF]. More recently, bats have also been identified as potential reservoirs of leptospires in a number of locations (e.g. [START_REF] Matthias | Diversity of bat-associated Leptospira in the Peruvian Amazon inferred by bayesian phylogenetic analysis of 16S ribosomal DNA sequences[END_REF][START_REF] Tulsiani | The role of fruit bats in the transmission of pathogenic leptospires in Australia[END_REF], with sometimes very high infection rates [START_REF] Lagadec | Pathogenic Leptospira spp. in bats, Madagascar and Union of the Comoros[END_REF]. Mathematical modeling has suggested seasonality of host reproduction as an important factor affecting Leptospira infection dynamics in rodent populations [START_REF] Holt | A model of Leptospirosis infection in an African rodent to determine risk to humans: seasonal fluctuations and the impact of rodent control[END_REF], however, no published information is currently available concerning Leptospira infection dynamics and maintenance in bat populations.

The Paramyxoviridae form a large virals family including the causative agents of many human and animal diseases, and some that have recently been linked to emerging and reemerging epidemics (e.g. Hendra and Nipah viruses; [START_REF] Aguilar | Emerging paramyxoviruses: molecular mechanisms and antiviral strategies[END_REF]. Rodents and bats have been recently shown to host a broad spectrum of paramyxoviruses [START_REF] Drexler | Bats host major mammalian paramyxoviruses[END_REF][START_REF] Wilkinson | Identification of novel paramyxoviruses in insectivorous bats of the Southwest Indian Ocean[END_REF][START_REF] Baker | Novel, potentially zoonotic paramyxoviruses from the African straw-colored fruit bat Eidolon helvum[END_REF]. Phylogenetic reconstruction of host/virus associations suggests frequent spillover events between different hosts, with a predominance of host shifts from bats to other animal species [START_REF] Drexler | Bats host major mammalian paramyxoviruses[END_REF][START_REF] Wilkinson | Major paramyxovirus exchange between introduced and endemic small mammals in the southwestern Indian Ocean region[END_REF], which has elsewhere been linked with the bat reproduction period [START_REF] Plowright | Reproduction and nutritional stress are risk factors for Hendra virus infection in little red flying foxes (Pteropus scapulatus)[END_REF]. For instance, [START_REF] Plowright | Reproduction and nutritional stress are risk factors for Hendra virus infection in little red flying foxes (Pteropus scapulatus)[END_REF] have identified reproduction as a key factor in the risk of Hendra virus infection in Australian fruit bats and especially in the emergence of these viruses in horse populations.

In this study, we investigated the dynamics of two pathogens of medical importance, the bacteria Leptospira and Paramyxoviruses, which have previously been described in insectivorous bats [START_REF] Lagadec | Pathogenic Leptospira spp. in bats, Madagascar and Union of the Comoros[END_REF][START_REF] Wilkinson | Identification of novel paramyxoviruses in insectivorous bats of the Southwest Indian Ocean[END_REF]. The tropical and endemic bat species Mormopterus francoismoutoui is the most abundant bat species on Réunion Island, and roosts in large colonies in a variety of sites such as bridges, houses, churches and caves.

We chose to study the largest known cave-based maternity colony of these bats in order to (i) determine whether the infection rate and/or the genetic diversity of infectious agents are influenced by host crowding and the succession of different developmental stages (adults, newborns and juveniles) over the breeding season, and (ii) compare the temporal infection profiles of both infectious agents. We hypothesize that transmission is host-density dependent favored by colony-induced crowding and fueled by the input of immunologically naïve juveniles [START_REF] Drexler | Amplification of emerging viruses in a bat colony[END_REF]. Results are expected to reveal specific characteristics of the ecology and evolution of Leptospira and paramyxoviruses and to highlight general patterns in pathogen maintenance in bat populations.

Results

Bat colony dynamics

The bat population studied was strictly composed of adults at the beginning of the survey in late November (Fig. 1a). A large number of newborns were observed in early January, together with the first juveniles (~3 weeks old), indicating that the birth pulse occurred from mid-December to mid-January. We estimated that the parturition period lasted up to mid-February as the last observation of newborns was made on February 8 th . Over all outings, a total of 2652 urine droplets were counted on the 16 plastic trays. The spatial distribution of the urine droplets suggested that bats use a main route for exiting the cave: those trays facing the center of the entrance were more exposed to bat passage than the ones placed on each side of the main flyway (χ 2 15 = 49.26, p < 0.001; model 1 in Table S3).

Trays lying directly underneath the main bat flyway (yellow-red in Fig. S1b) showed statistically significant variations in the number of urine droplets collected over time (χ 2 7 = 48.43, p < 0.001; Fig. 1b and model 1 in Table S3). Indeed, at the first sampling session (i.e.

November 20 th ), the number of exiting adult bats reached a maximum: 46 500 individuals as estimated by the video. Following this maximum, the number of exiting bats declined drastically to reach a minimum on January and remained very low for about three additional weeks. This corresponds to the parturition period where females gave birth and thus remained with newborns leading to this reduced pattern of exit. Then, the number of exiting bats increased to reach a second peak on mid-February, coinciding with the first flights of juvenile bats. Beyond that time point, the number of exiting bats declined progressively towards the end of the season corresponding to the definitive emptying of the cave; the number of exiting bats estimated by video analysis was still 13 400 individuals at the end of the sampling period (i.e. March 21 st ). In late May, a last visual inspection showed that the cave was unoccupied.

Leptospira and paramyxovirus infection dynamics

Altogether, 420 individual urine droplets were screened by PCR for Leptospira and paramyxoviruses during the 2012-2013 breeding season. The bacterial and viral excretion positively identified individual bats as being currently infected with either of the two infectious agents. Hereafter, "infection rate" refers to the proportion of PCR-positive samples.

Both Leptospira and paramyxovirus infection rates varied significantly over the breeding season (Leptospira: χ 2 7 = 25.209, p < 0.001; paramyxovirus: χ 2 7 = 59.879, p < 0.001, models 2 and 3 in Table S3) and revealed similar patterns of temporal variation (Fig. 1c). The relative intensity of Leptospira excretion also showed significant variation over time, despite the large variation in the Ct values (χ 2 7 = 148.06, p = 0.003, fig. 1d and model 4 in Table S3). Overall, infection dynamics were characterized by two peaks, both in the rates of infection (Fig. 1c)

and the intensity of Leptospira excretion (Fig. 1d). Notably, the first peak occurred at the beginning of the breeding season, during the late-pregnancy period of females, with infection rates (±95% confidence interval) reaching 45% (±12%) and 61% (±11%) for Leptospira and paramyxovirus, respectively. Later, during the parturition period, both infection rates (as well as the intensity of Leptospira excretion) decreased drastically down to a minimum of 6% (±6%) and 4% (±5%) for Leptospira and paramyxovirus respectively (Table S4). The second infection peak (as well as the intensity of Leptospira excretion) occurred roughly two months after the beginning of the parturition period. Towards the end of the season, infection rates slightly decreased, with Leptospira and paramyxovirus detection rates reaching 21% (±11%) and 31% (±13%), respectively. Infection rates were also monitored at the start of the following season in order to collect data on the early stages of colony formation. Infection rates were 28% (±16%) and 21% (±16%) on October 18 th 2013 for Leptospira and paramyxovirus respectively, and increased within 2 weeks, to reach 62% (±18%) and 52% (±18%) respectively (Table S4).

Our data showed that bats were frequently co-infected (Fig. 2a). Statistically, the presence of one infectious agent was strongly correlated to the presence of the other agent (χ 2 1 = 22.170, p < 0.001, models 2 and 3 in Table S3). Moreover, the rate of co-infection significantly varied over the breeding season (χ 2 7 = 41.761, p < 0.001) and followed the same dynamics as mono-infections (Fig. 2b and model 5 in Table S3). However, the expected probability of being co-infected at random for each sampling date was not significantly different from the observed values (G-test: p > 0.1 for all tests; Fig. 2b). Finally, the intensity of Leptospira excretion was not affected by co-infection with paramyxovirus (χ 2 1 = 0.020, p = 0.957, model 4 in Table S3).

Genetic diversity of Leptospira and paramyxovirus

Phylogenetic analyses of Leptospira provided similar results for secY and rrs2 genes (Fig. 3a and Fig. S4 respectively). Both phylogenetic trees revealed the circulation of a single haplotype in the maternity colony, closely related to Leptospira borgpetersenii. This haplotype was distinct from those previously described in wild animals and humans in the western Indian Ocean. In contrast, paramyxoviruses detected in the cave were much more diverse as 4 different genetic groups were observed (Fig. 3b). The first group corresponds to virus sequences associated with three different bat families of the western Indian Ocean region (Molossidae, Miniopteridae and Verspertilionidae). The second group includes virus sequences detected in the very closely related bat species M. acetabulosus (Mauritius) and M. jugularis (Madagascar). Virus sequence in the third group were initially identified in kidney tissue from a rat caught in Réunion Island near the studied cave [START_REF] Wilkinson | Major paramyxovirus exchange between introduced and endemic small mammals in the southwestern Indian Ocean region[END_REF].

Finally, one sequence grouped with viruses largely associated with rats in the region (group 4). There was no evidence of any correlation between the occurrence of the different Paramyxovirus genetic groups and sampling date (p = 0.467), suggesting that the 4 genetic groups were likely co-circulating within the colony.

Discussion

Previous studies have demonstrated that dynamics of bat-hosted infectious agents exhibit a strong seasonal pattern, with infection peaks being recorded during the breeding period [START_REF] Turmelle | Ecology of Rabies Virus Exposure in Colonies of Brazilian Free-Tailed Bats (Tadarida brasiliensis) at Natural and Man-Made Roosts in Texas[END_REF][START_REF] Drexler | Amplification of emerging viruses in a bat colony[END_REF]. Here, using a non-invasive sampling strategy in a natural maternity colony, we monitored the excretion of both a bacterial and a viral agent, namely Leptospira and paramyxovirus, by a tropical insectivorous bat species endemic to Réunion Island. Our data provide evidence that infection rates express strong temporal fluctuations within the time scale of the breeding season. We find that Leptospira and paramyxoviruses display remarkably comparable infection dynamics, a feature suggestive of synchronized host susceptibility and a common transmission route within the colony, i.e.

urine. Data from two successive breeding seasons showed epidemic bursts occurring during colony formation and two-months after the birth pulse. These findings strongly suggest the occurrence of a vigorous horizontal transmission within the maternity colony and support that seasonal coloniality and synchronized bat behavior is a major determinant of infection dynamics at the population level.

2011).

Following the infection burst that occurs early-on in colony formation, a rapid and highly synchronous decline in Leptospira and paramyxovirus excretion was observed, suggesting that a large majority of adult bats rapidly control the acute infection and that excretion of infectious agents is only transitory. This pattern is most likely the consequence of the synchronous development of a robust immune response in bats against the two infectious agents. Such an immune response would not only protect parturient females, but also newborns through the passive transfer of specific antibodies from mother to offspring as demonstrated experimentally in frugivorous bat species [START_REF] Baker | Viral antibody dynamics in a chiropteran host[END_REF][START_REF] Epstein | Duration of Maternal Antibodies against Canine Distemper Virus and Hendra Virus in Pteropid Bats[END_REF], and largely suggested by several field studies [START_REF] Plowright | Reproduction and nutritional stress are risk factors for Hendra virus infection in little red flying foxes (Pteropus scapulatus)[END_REF][START_REF] Breed | Evidence of endemic Hendra virus infection in flying-foxes (Pteropus conspicillatus)--implications for disease risk management[END_REF], including one carried out on insectivorous bats [START_REF] Drexler | Amplification of emerging viruses in a bat colony[END_REF]. Though transitory, the conferred passive protection may shape age related patterns of infection [START_REF] Boulinier | Maternal transfer of antibodies: raising immunoecology issues[END_REF] and has been suggested to last between one and six months after maternal severing in insectivorous [START_REF] Drexler | Amplification of emerging viruses in a bat colony[END_REF] and frugivorous bats [START_REF] Baker | Viral antibody dynamics in a chiropteran host[END_REF][START_REF] Epstein | Duration of Maternal Antibodies against Canine Distemper Virus and Hendra Virus in Pteropid Bats[END_REF], respectively. Thus, the influx of juveniles in the group of susceptible bats is delayed until the disappearance of passively transferred maternal antibodies. Indeed, the two-months delay observed before appearance of the second infection peak is consistent with such waning of passively transferred maternal antibodies, as previously suggested by a field study on Myotis bats [START_REF] Drexler | Amplification of emerging viruses in a bat colony[END_REF]. In addition to an immunization of the mothers, the emergence of uninfected juveniles may contribute to the prevalence decrease measured in February. However, considering that females give birth to a single pup, this decrease should be at maximum of 50%, which is consistently lower than the actual observed drop (i.e. xx% for Leptospira and xx% for paramyxovirus, respectively).

Thus we propose that the dramatic decrease in prevalence results from both immunization of the mothers and the emergence of transiently immunized juveniles. Interestingly, some of the expected positive relationship between host density and infection prevalence [START_REF] Kallio | Hantavirus infections in fluctuating host populations: the role of maternal antibodies[END_REF] may be lacking at the end of the season when host density has increased by twice with the birth of newborns. The number of immune females may explain why infection rates, and especially that of paramyxovirus, did not return to the peak levels measured at the beginning of the season. Further investigations addressing the serological status of pregnant mothers, newborns and juveniles towards Leptospira and paramyxovirus need to be carried out in order to properly address these major points.

Seasonality of reproduction might play a role in the evolution of infectious agents by causing alternating periods of potential mixing of different lineages with high transmission and population bottlenecks that simultaneously limit strain diversity and cause rapid genetic shifts (e.g. [START_REF] Ferguson | Ecological and immunlogical determinants of influenza evolution[END_REF]. In the present study, we observed that one single haplotype of Leptospira co-circulated with multiple paramyxovirus lineages, though no apparent evolution of the viral diversity could be recorded over the timescale of our observations.

Multiple circulating virus strains may affect within-host and within population dynamics by conferring cross-immunity [START_REF] Hayman | Ecology of zoonotic infectious diseases in bats: current knowledge and future directions[END_REF]. Interestingly, we also found evidence of paramyxovirus sharing between bat and rodent populations, suggesting that host shift between rats and bats may contribute to the paramyxovirus diversity observed within the maternity colony.

The presence of multiple infectious agents within a host, i.e. co-infection, can critically impact infection dynamics [START_REF] Telfer | Species interactions in a parasite community drive infection risk in a wildlife population[END_REF]. Interactions may be synergistic or antagonistic and infectious agents may interact either directly or indirectly via "bottom-up" (e.g. competition for shared host resources) or "top-down" processes (e.g. immune-mediated competition or facilitation) [START_REF] Pedersen | Emphasizing the ecology in parasite community ecology[END_REF]. Here, although bats were frequently co-infected with Leptospira and paramyxovirus, the observed rate of co-infection was not different from what is expected at random and we found that the presence of paramyxovirus infection had no effect on the intensity of Leptospira excretion. The possibility that the intensity of paramyxovirus infection could be positively influenced by Leptospira infection could not be tested with our experimental setup and, thus, cannot be ruled out. The large confidence intervals for pathogen prevalence and intensity of Leptospira infection preclude any solid conclusion but our study suggest that co-infection most likely appears as the mere consequence of the temporal conjunction between the infection dynamics and did not result in any apparent interaction between the two pathogens.

In conclusion, our study has focused on infection dynamics within the time frame of the bat breeding season. Although it is difficult to distinguish between episodic shedding from persistently infected bats and transient epidemics [START_REF] Plowright | Ecological dynamics of emerging bat virus spillover[END_REF], our result suggest that following epidemic bursts, the vast majority of infected bats likely develop immunity efficiently and stop excreting pathogens; however, the question of infection maintenance beyond the breeding season has not been addressed. A recent theoretical study, using stochastic epidemiological models with a seasonal birth pulse, suggests that pre-existing immunity is critical for pathogen maintenance in bat populations [START_REF] Peel | The effect of seasonal birth pulses on pathogen persistence in wild mammal populations[END_REF]). Here, rates of infection at the beginning and end of the breeding season were roughly similar (~25%) implying that the bat maternity in fine serves as an ecological "vaccination center", providing potential antibody immunity to large proportions of the population outside of the breeding season. Transmission outside of maternity colonies may thus be maintained by a small fraction of individuals chronically excreting the two pathogens, waning immunity and/or immigration events between colonies [START_REF] Breed | Evidence of endemic Hendra virus infection in flying-foxes (Pteropus conspicillatus)--implications for disease risk management[END_REF][START_REF] Field | Hendra virus infection dynamics in Australian fruit bats[END_REF][START_REF] Sohayati | Evidence for Nipah virus recrudescence and serological patterns of captive Pteropus vampyrus[END_REF][START_REF] Peel | Henipavirus neutralising antibodies in an isolated island population of African fruit bats[END_REF]. As fission-fusion social structures are being increasingly recognized in bats [START_REF] Kerth | Bats are able to maintain long-term social relationships despite the high fission-fusion dynamics of their groups[END_REF], and because the bat species studied herein tends to split into small populations during non-reproductive period, it is possible that a complex meta-population structure will influence seasonal infection dynamics within the community [START_REF] Plowright | Urban habituation, ecological connectivity and epidemic dampening: the emergence of Hendra virus from flying foxes (Pteropus spp.)[END_REF]. Multidisciplinary research on the interplay between population structures and temporal dynamics in the context of their associated pathogens are important for the prediction of possible emergence events in specific geographic regions [START_REF] Restif | Model-guided fieldwork: practical guidelines for multidisciplinary research on wildlife ecological and epidemiological dynamics[END_REF], such as the western Indian Ocean, where leptospirosis and paramyxoviruses are widely prevalent [START_REF] Bourhy | Isolation and characterization of new Leptospira genotypes from patients in Mayotte (Indian Ocean)[END_REF][START_REF] Lagadec | Pathogenic Leptospira spp. in bats, Madagascar and Union of the Comoros[END_REF][START_REF] Wilkinson | Identification of novel paramyxoviruses in insectivorous bats of the Southwest Indian Ocean[END_REF][START_REF] Desvars | Endemicity of leptospirosis in domestic and wild animal species from Reunion Island (Indian Ocean)[END_REF][START_REF] Dietrich | Diversification of an emerging pathogen in a biodiversity hotspot: Leptospira in endemic small mammals of Madagascar[END_REF].

Experimental procedures

Urine collection

A colony of the insectivorous bat Mormopterus francoismoutoui was monitored during the 2012-2013 breeding season, in Réunion Island, a tropical oceanic Island located 800km East of Madagascar. The maternity colony is sited in a natural cave of approximately 30m 3 and is occupied by one single bat species, i.e. M. francoismoutoui, from October to May. The maternity colony is mostly composed of adult females at the early stages of the breeding season. A total of eight field outings were carried out from November 2012 to April 2013, a timing that did not allow monitoring the first weeks of the colony formation. We hence conducted additional monitoring in the same cave during the next season in the very early stages of colony formation, with four field outings during October 2013. A non-invasive sampling strategy was implemented in order to avoid direct manipulation of bats thus limiting disturbance of the colony. This strategy is based on the demonstration that infected bats excrete both Leptospira and paramyxovirus in their urine [START_REF] Baker | Co-circulation of diverse paramyxoviruses in an urban African fruit bat population[END_REF][START_REF] Desvars | Endemicity of leptospirosis in domestic and wild animal species from Reunion Island (Indian Ocean)[END_REF]. In addition, urine droplets are spontaneously shed by flying bats during their daily emergence at dusk and could then be collected on plastic films layered on the ground. For each sampling date, 16 plastic film (Saran wrap) -covered cardboard rectangles (20x15cm)

were placed inside larger plastic trays and positioned at the entrance of the cave following a referenced map. This allowed positioning each tray at the exact same place throughout the whole survey. As shown on the overview of the sampling design provided in Fig. S1 trays were separated by at least 1 meter in order to minimize the possibility of sampling several urine droplets from a single bat specimen. After the emergence of bats, up to 5 distant (about 5cm) urine droplets assumed to correspond to 5 independent bat specimens, were collected from each of the 16 plastic trays and buffered in micro-tubes containing 300 µL of Minimum Essential Medium Eagle buffer. Samples were then kept in the field in a cool box filled with ice packs and brought back to the lab where they were immediately stored at -80°C until tested.

Bat colony dynamics

The colony was visually monitored throughout the whole study in order to estimate the population age structure and timing of parturition. The presence of adults (brown), newborns (pink colored) and juveniles (dark grey) was thus checked by visual inspection at each sampling date (see photos in Fig. S2).

We attempted to characterize changes in bat behavior during the breeding season by monitoring the numbers of bats at emergence. Relative estimates were obtained by counting the number of individual urine droplets landing on the plastic films, as we showed that the number of urine droplets correlates with the number of exiting bats (see details in Fig. S3).

Thus, collected figures allowed comparative analysis between sampling field missions.

Video analyses were also used to estimate the absolute size of the exiting population.

When lightness and weather conditions permitted, videos of bats leaving the cave were acquired using the sky as a backdrop. Custom MATLAB-encoded algorithms were then used to define a line within the video-images and video frames were segmented based on the contrast of each image (dark bats on a clear sky background). A binary image was then constructed using mask data along the defined line (X-dimension), and for each frame of the video (Y-dimension). Non-touching objects were then counted in the obtained image mask to generate a minimum-number estimate of the number of bats that had left the cave.

Screening and genetic diversity of Leptospira and Paramyxovirus

Paramyxoviruses are single stranded negative sense RNA viruses, and thus their nucleic acids require reverse transcription before detection via PCR. Therefore, detection was carried out on a single cDNA preparation for both bacterial and viral detection. Using QIAamp Mini spin columns (Qiagen, Valencia, CA), total nucleic acids were extracted following manufacturer"s instructions from 140µl of each buffered urine sample. A negative control was added for each extraction run. Following extraction, 10 µL of total nucleic acids were submitted to reverse transcription using the Promega cDNA kit (Promega, Madison, USA) with 1.25 µL of Hexamers (Promega, Madison, USA), following manufacturer"s instructions.

Five microliters of cDNA were then used as templates for Leptospira and paramyxovirus detections using previously described detection methods [START_REF] Smythe | A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp[END_REF][START_REF] Tong | Sensitive and broadly reactive reverse transcription-PCR assays to detect novel paramyxoviruses[END_REF]. Briefly, the presence of Leptospira was detected using a probe-specific real-time PCR, targeting a fragment of the 16S rRNA gene, specific of all known pathogenic Leptospira species. The threshold cycle (Ct) of positive samples was noted to infer the relative bacterial load and thus the relative intensity of Leptospira excretion. In order to compare Ct values among different PCR runs, we verified the consistency of the Ct value for the positive control in each run. For positive samples, a partial fragment of the 16S rRNA and secY genes was then amplified, as described in [START_REF] Dietrich | Diversification of an emerging pathogen in a biodiversity hotspot: Leptospira in endemic small mammals of Madagascar[END_REF], and sequenced (Genoscreen, Lille, France)

for subsequent phylogenetic analyses. For the detection of paramyxoviruses, a semi-nested PCR targeting the polymerase-encoding gene was performed as previously described [START_REF] Wilkinson | Identification of novel paramyxoviruses in insectivorous bats of the Southwest Indian Ocean[END_REF]. After electrophoresis on a 2% agarose gel stained with GelRed (Biotium Inc.), PCR products of the approximate anticipated size (450-500 bp) were purified using the QIAGEN PCR purification kit, cloned into the pGEMt vector system (Promega), and submitted to direct Sanger sequencing (Genoscreen, Lille, France). DNA sequences of paramyxoviruses obtained from independent bacterial clones were aligned to generate consensus sequences for each sample in order to correct for the majority of sequencing or PCR-introduced errors. These sequence data have been submitted GenBank database under accession number KJ607934-KJ607957 for Leptospira and KJ748495-KJ748520 for paramyxoviruses Nucleotide sequences were assembled and edited manually using ChromasLite 2.01 (Technelysium Pty Ltd, South Brisbane, Australia), and aligned using CLC Sequence Viewer 6.8 (CLC bio, Aarhus, Denmark) with references sequences including samples from bats and terrestrial small-mammals of the western Indian Ocean Islands (see details in Tables S1 andS2). Phylogenetic trees were constructed using PHYML [START_REF] Guindon | A Simple, Fast, and Accurate Algorithm to Estimate Large Phylogenies by Maximum Likelihood[END_REF] under the AIC best-fitted model of evolution (Leptospira: TPM3uf+I and HKY+I for 16S rRNA and secY; Paramyxoviruses: TIM3+I+G) selected by JMODELTEST v.0.1.1 [START_REF] Posada | jModelTest: phylogenetic model averaging[END_REF], a BIONJ as starting tree and NNI tree search. The robustness of branches was evaluated performing bootstrap analysis with 1000 repetitions.

Statistical analyses

We examined changes in the number of urine droplets (as a correlate of the number of bats emerging at dusk) over the breeding season and among the trays by using a Generalized Linear Model (GLM) with a Poisson distribution and a log link function. "Sampling date" and "tray number" were included as explanatory variables.

We investigated the dynamic of infections (both mono-and co-infections) by using GLMs with binomial error and a logit link function, with the "sampling date" as an explanatory variable. We aimed to determine whether the presence of one infectious agent was associated with the presence of the alternative infectious agent, adding "infection status"

for Leptospira and paramyxoviruses as explanatory variables in models testing mono-

  Synchronized host susceptibility in females may have resulted from a transient pregnancy-related depressed immunity. Increased susceptibility to infectious agents during pregnancy has been demonstrated in different mammalian systems (e.g.[START_REF] Sheldon | Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology[END_REF][START_REF] Cattadori | Peak shift and epidemiology in a seasonal host-nematode system[END_REF]) and short-term trade-offs between immune activity and reproduction have been suggested to explain such patterns. In the greater mouse-eared bat (Myotis myotis), for instance, weaker cell-mediated responses and heavier ectoparasite infestations have been observed in pregnant females compared to non-reproductive females from the same roost[START_REF] Christe | Variation in intensity of a parasitic mite (Spinturnix myoti) in relation to the reproductive cycle and immunocompetence of its bat host (Myotis myotis)[END_REF]. Our sampling design does not give access to the immunological status of the population, and thus does not allow verification of this hypothesis. However, such undulating patterns of immunomodulation and viral replication in females during late-pregnancy have already been reported in other field studies investigating Hendra or filo-viruses dynamics in frugivorous bats (e.g.[START_REF] Plowright | Reproduction and nutritional stress are risk factors for Hendra virus infection in little red flying foxes (Pteropus scapulatus)[END_REF][START_REF] Breed | Evidence of endemic Hendra virus infection in flying-foxes (Pteropus conspicillatus)--implications for disease risk management[END_REF]

Fig. 1 .

 1 Fig. 1. Seasonal dynamics of (a, b) the bat population and (c, d) Leptospira and

Fig. 2 .

 2 Fig. 2. (a) Proportion of mono-and co-infections among infected individuals and (b) seasonal

Fig. 3 .

 3 Fig. 3. Phylogenetic relationships of (a) Leptospira and (b) paramyxovirus detected in the

Acknowledgements

We are grateful to Dominique Bang for kindly giving us access to the bat maternity colony, and to Marina Béral, Lisa Cavalerie, Yann Gomard, Camille Lebarbenchon, Gildas Le Minter, and Julien Mélade for their help in field sampling. We thank Audrey Jaeger and Olivier Flores for advice on statistical analyses. This work was supported by the CPER/ Regional council/European Regional Development Funds ERDF-POCT; Réunion, LeptOI (N°32913), FSOI (N°31189) and ParamyxOI (N°33857) projects, granted to PT, KD and DAW respectively. The postdoctoral fellowships of MD and DAW were funded by "RUN-Emerge: A Regpot European project funded by European Commission under FP7 program". BR received postdoctoral grants from RunEmerge project, from "Fonds de Coopération Régionale" of the Préfecture de La Réunion and from the Dr. Ralph and Marian Falk Medical Research Trust to The Field Museum of Natural History, Chicago. The authors declare no conflict of interest.

infections. A G-test was used to test differences between the co-infection rates, as observed or as expected at random. Expected co-infection rates at each sampling date were calculated as the product of the rates of infection with each single pathogen measured at the same date [START_REF] Nieto | Meta-analysis of coinfection and coexposure with Borrelia burgdorferi and Anaplasma phagocytophilum in humans, domestic animals, wildlife, and Ixodes ricinus-complex ticks[END_REF].

The temporal variation in the relative intensity of pathogen excretion in urine was analyzed for Leptospira only, as no accurate quantitative information on viral load could be obtained from the semi-nested PCR used for paramyxovirus detection. We used a GLM with a Gaussian distribution and a logit link function, using the threshold cycle (Ct) as a proxy for the relative intensity of Leptospira excretion and "sampling date", "tray number" and "paramyxovirus infection status" as explanatory variables.

All starting models were simplified by backward stepwise elimination of non-significant terms (p > 0.05) beginning with interactions, to obtain the minimum adequate model [START_REF] Crawley | The R Book John Wiley[END_REF] (see models in Table S3). In models using a binomial error structure, we systematically checked for over dispersion by calculating that the ratio of residual deviance over residual degrees of freedom was <2. Curves were produced using a loess smoother.

Evolution of pathogen genetic diversity (proportion of the different lineages over time) was analyzed using a Fisher"s exact test. All analyses were carried out using the R software package v.3.0.2. accession numbers are indicated in parentheses. Bootstrap values higher than 80% are represented by a dark circle.