Ani Hsieh

Simon Lacroix

Editorial for the JFR special issue on "Multiple Collaborative Field Robots"

Editorial for the JFR special issue on "Multiple Collaborative Field Robots" Ani Hsieh and Simon Lacroix July 11, 2012 The deployment of multiple robots can lead to important operational benefits in many field applications, for either exploration, surveillance and intervention missions. With respect to a single robot, teams of robot are obviously less prone to failures, able to simultaneously operate over larger areas and to convey a more and larger variety of payloads. But deploying multiple robots also yields benefits to the robots themselves: the robotics literature has already proposed numerous multi-robot schemes in which robots not only cooperate to achieve a given mission, but assist each other to palliate encountered difficulties. Robots can act as communication relays, locate others or merge maps to improve the spatial consistency of maps, carry smaller robots, evolve information to ensure safer or quicker motions, etc.

Several contributions of multiple field robot systems have been proposed in a former special issue of the Journal of Field Robotics in 20071 . Research on such systems has much evolved since, and in 2010 an ambitious challenge has been set up in Adelaide, Australia, in which several teams competed to achieve a surveillance mission with more than a dozen of robots. Six articles of this special issue report on this Multi-Autonomous Ground-robotic International Challenge (MAGIC) sponsored by the The Defence Science & Technology Organization (DSTO) in Australia and the Research Development & Engineering Command (RDECOM) in the United States of America. The MAGIC 2010 contributions highlight the technical challenges in planning, perception, and command and control that has been overcome to operate successfully in the field. In contrast, the last two contributions of this Special Issue report on field experiments conducted with a team of field robots in environments that were directly and indirectly damaged by the 2011 Tohoku earthquake in Sendai, Japan.

To lay the foundation for the MAGIC 2010 competition, Finn et al. depict the metrics defined to assess the performance of the MAGIC competitors. After a summary of the scenarios and missions in which numerous heterogeneous robots have to detect an "neutralize" static and mobile targets in a large scale rurban environment, the article depicts the various criteria used to measure the performance of the robot teams. The task is not an easy one, as mission-level operational criteria have to be mixed with systems level criteria, that include human robot interactions, and technological criteria pertaining to single robots. The robotics community is paying more and more attention to evaluation metrics, which are naturally essential to assess robotics systems: here a thorough work on evaluating robot teams interacting with operators in a realistic scenario is proposed -and has been exploited to rank the MAGIC competitors.

With the rules clearly outlined, the issue segues into the various teams' efforts. While different approaches were taken, common themes emerge. For one, the use of commercial off-the-shelf components as the foundation of each team's effort coupled with the development of a unifying system architecture. Boeing et al. outline team MAGICian's approach towards developing a unified system architecture with the necessary sensing, planning, navigation, and mapping capabilities needed to address the competition's demands. Key to their success was an agile development methodology that took advantage of commercial off-the-shelf hardware and software components which enabled the team to focus on system integration. A similar approach taken by team RASR as described by Lacaze et al. In these two contributions, a common theme is the use of commercial off-the-shelf hardware and software components to minimize risk in the design phase and to identify focus areas. In addition, team RASR incorporated a distributed coordination strategy which focuses on minimizing communications between the various robots.

A second theme that emerges is the emphasis on the human and robotic team command and control interface. Butzke et al. describe the University of Pennsylvania entry in MAGIC. The article presents how the authors engineered a hierarchical architecture to interleave mapping, localization, exploration and target management processes within each UGV, and emphasizes the way two operators can operate a team of nine robots endowed with this architecture. Olson et al. describe their teams approach towards developing the winning MAGIC 2010 system. In this article, Team Michigan presents their general design approach which focused on minimizing the efforts required by the human operators when commanding and controlling a team of fourteen autonomous vehicles. Specifically, the team describes their ability to maintain a consistent global map by developing fast loop-closing, map optimization, and communications algorithms. These developments, in conjunction with a centralized planning architecture that enabled robots to execute their individual tasks in a centrally coordinated fashion, were the key to their success.

A third and common theme is the need for a careful management of communications. Guivant et al. describe their team's approach which centered on the development of the interface between the communication and processing modules to achieve a flexible control architecture that can support traditional teleoperation to point-and-click autonomy. In this work, the authors describe how careful management of the available system bandwidth enabled their teams to achieve long-range communication while still providing real-time map data.

If one views the MAGIC 2010 contributions as a celebration of the recent achievements in the field of collaborative robotic systems, the two last contributions should be viewed as challenges that must be overcome. Murphy et al.

reports on the experience of two deployments of heterogeneous unmanned marine vehicle teams at the 2011 Great Eastern Japan Earthquake response and recovery by the Center for Robot-Assisted Search and Rescue (USA) in collaboration with the International Rescue System Institute (Japan). This work reports on the teams successes and failures. Specifically, Murphy et al. showed how their planned multi-robot systems did not fall into traditional taxonomies and the resulting challenges. Lastly, Michael et al. reports on their deployment of a team of aerial and ground robots for collaborative mapping of an earthquake-damaged building. Specifically, the team reports on the experiments that were conducted in a structurally compromised building at Tohoku University in Sendai, Japan that was damaged during the 2011 Tohoku earthquake. The team shows the ability of the team of heterogeneous robots in providing 3-D maps of the building and discuss future challenges from their experiences.

It has been a great pleasure working with all the contributing authors and the numerous reviewers in bringing together this special issue. In many ways, this issue is a small microcosm of the various developments in the field of collaborative field robotic systems. We hope you will enjoy these articles as much as we have.

Journal of Field Robotics, special Issue on Teamwork in Field Robotics. Volume

24, Issue 11-12, Nov. -Dec. 2007


Rime Sioud

Riadh Khalifa

Nicolas Houel
email: nicolas.houel@eso-suposteo.fr

Olivier Caporossi

Serge Pin

Laurent Stubbe

Jeanine Sanchez

Auditory cues behind congenitally blind subjects improve their balance control in bipedal upright posture

Keywords: congenital blindness, balance control, auditory signal, bipedal, unipedal

Background: Congenitally blind subjects developed postural adaptations improving somatosensory and vestibular systems to maintain upright stability and auditory skills to orient them in environment. However, the influence of auditory cues on upright stability in congenitally blind subjects stays unknown.

Research question: The aim of this study is to define the influence of an auditory cue in congenitally blind subjects back space on their balance posture.

Methods: Eleven sighted subjects and eleven congenitally blind subjects performed upright bipedal and unipedal quiet stances on a force plate with two conditions of auditory cue played by a loudspeaker placed 2 meters behind them. Mean CoP velocity were recorded. Student test was used to compare significant difference between blind and sighted subjects bipedal and unipedal postures stability in both conditions of auditory cue.

Results and significance: Results showed that congenitally blind subjects had no significant difference in mean sway velocity compared to sighted subjects in bipedal upright posture in auditory signal condition. However, blind subjects had significant lower mean sway velocity than sighted subjects in bipedal upright posture without sound. Blind subjects had significant increased mean sway velocity during unipedal quiet standing in both auditory cue conditions (with and without sound). The results showed that congenitally blind subjects used auditory cues placed behind them in order to improve their balance control in bipedal upright posture. In this case, blind subjects could better use compensatory mechanisms to perform quiet standing as sighted subjects. Without sound or in unipedal upright posture, congenitally blind subjects probably have sensory perturbations or limitations that impose them adaptations in order to avoid falling risk. Auditory cues should be study in the aim to better understand the compensatory mechanisms used by congenitally blind subjects to perform postural balance in usual environment.

Introduction

Predictions about total blindness prevalence could reach about 39 millions of people all over the world in 2020 [START_REF] Ackland | World blindness and visual impairment: despite many successes, the problem is growing[END_REF]. For these people, various day life activities, like opening a door or waiting at crosswalk, need to maintain upright posture according to external constraints imposed by environment and without visual information. In this case, upright posture with visual impairment improve falling risk [START_REF] Horak | Postural orientation and equilibrium: what do we need to know about neural control of balance to prevent falls?[END_REF][START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF][START_REF] Giagazoglou | Static balance control and lower limb strength in blind and sighted women[END_REF]. Because vision has major importance in balance control during quiet standing [START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF][START_REF] Giagazoglou | Static balance control and lower limb strength in blind and sighted women[END_REF][START_REF] Massion | Movement, posture and equilibrium: interaction and coordination[END_REF][START_REF] Winter | Human balance and posture control during standing and walking[END_REF][START_REF] Alotaibi | Effect of absence of vision on posture[END_REF], various studies tried to describe upright posture center of pressure (CoP) fluctuation and body sway in blind subjects [START_REF] Alotaibi | Effect of absence of vision on posture[END_REF]. Blind subjects adopt postural strategies with modified perception of environment [START_REF] Alotaibi | Effect of absence of vision on posture[END_REF][START_REF] Grivas | Prevalence of scoliosis in women with visual deficiency[END_REF]. Visual impairment could be partly compensated by vestibular and somatosensory systems in order to maintain balance posture [START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF][START_REF] Soares | Postural control in blind subjects[END_REF]. People with congenital or more than three years blindness developed postural control adaptations that could make up absence of vision [START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF][START_REF] Soares | Postural control in blind subjects[END_REF][START_REF] Rougier | Adaptative effects of loss of vision on upright undisturbed stance[END_REF]. As example, congenitally blind subjects have better balance control in bipedal quiet standing on different supports (solid and foam surfaces) than subjects with visual acquired impairment [START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF]. Congenitally blind subjects could compensate their visual impairment increasing the use of somatosensory and vestibular systems to maintain upright stability [START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF][START_REF] Rougier | Adaptative effects of loss of vision on upright undisturbed stance[END_REF][START_REF] Parreira | Postural control in blind individuals: A systematic review[END_REF].

Blind subjects in quiet standing present lower difference between center of gravity and CoP displacements [START_REF] Rougier | Adaptative effects of loss of vision on upright undisturbed stance[END_REF] and lower sway amplitude of their CoP than sighted subjects with closed eyes [START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF][START_REF] Rougier | Adaptative effects of loss of vision on upright undisturbed stance[END_REF]. This reduced CoP sway amplitude has been notably observed in congenitally blind subjects [START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF]. According to these studies, Blind subjects have developed motor organization where somatosensory system changes muscular stiffness in order to maintain stance without external or environmental perturbation [START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF][START_REF] Rougier | Adaptative effects of loss of vision on upright undisturbed stance[END_REF]. As consequence, blind subjects have faster reaction times to overcome balance control associated with ground platform perturbations compared to sighted subjects [START_REF] Nakata | Automatic postural response systems in individuals with congenital total blindness[END_REF]. Blind subject showed better proprioceptive acuity of ankle dorsi-flexion than sighted subjects [START_REF] Ozdemir | Sensorimotor posture control in the blind: superior ankle proprioceptive acuity does not compensate for vision loss[END_REF]. Blind subjects have similar muscle pattern activities compared to sighted subjects in various upright posture [START_REF] Nakata | Automatic postural response systems in individuals with congenital total blindness[END_REF]. In all cases, Blind subjects could maintain their postural balance without influence on their muscular lower limb's strength that remains similar to sighted subjects [START_REF] Giagazoglou | Static balance control and lower limb strength in blind and sighted women[END_REF]. As consequence, upright balance control in blind subject is nearby sighted subject with eyes closed in static and mobile ground platform [START_REF] Giagazoglou | Static balance control and lower limb strength in blind and sighted women[END_REF][START_REF] Schmid | Equilibrium during static and dynamic tasks in blind subjects: no evidence of cross-modal plasticity[END_REF]. As for sighted subjects with eyes closed, head and hip of blind subjects move together and follow floor's oscillations [START_REF] Schmid | Equilibrium during static and dynamic tasks in blind subjects: no evidence of cross-modal plasticity[END_REF][START_REF] Jeka | Haptic cues for orientation and postural control in sighted and blind individuals[END_REF]. By not exploiting all body's degrees of freedom, blind subjects use a strategy to increase the vestibular system stimulation which may be related to the presence of anxiety [START_REF] Schmid | Equilibrium during static and dynamic tasks in blind subjects: no evidence of cross-modal plasticity[END_REF][START_REF] Buchanan | Vestibular loss disrupts control of head and trunk on a sinusoidally moving platform[END_REF]. However, some results about blind subject's skills in order to overcome visual impairment during balance control remain unclear [START_REF] Schmid | Equilibrium during static and dynamic tasks in blind subjects: no evidence of cross-modal plasticity[END_REF]. As example, whereas blind subjects seems to develop specific haptic cues [START_REF] Parreira | Postural control in blind individuals: A systematic review[END_REF][START_REF] Nakata | Automatic postural response systems in individuals with congenital total blindness[END_REF][START_REF] Jeka | Haptic cues for orientation and postural control in sighted and blind individuals[END_REF], their head kept larger movements than sighted subjects when postural control was associated with additional haptic aids [START_REF] Jeka | Haptic cues for orientation and postural control in sighted and blind individuals[END_REF]. In some other studies, blind subjects present lower antero-posterior bipedal upright stability than sighted subjects with eyes closed [START_REF] Giagazoglou | Static balance control and lower limb strength in blind and sighted women[END_REF][START_REF] Ozdemir | Sensorimotor posture control in the blind: superior ankle proprioceptive acuity does not compensate for vision loss[END_REF]. Few studies tried to understand the respective influences of vestibular and sensorimotor skills in blind subjects in single leg stance upright posture [START_REF] Giagazoglou | Static balance control and lower limb strength in blind and sighted women[END_REF][START_REF] Ozdemir | Sensorimotor posture control in the blind: superior ankle proprioceptive acuity does not compensate for vision loss[END_REF]. For these studies, vestibular and sensorimotor skills can't compensate the absence of vision in unipedal quiet standing. That the reason why blind subjects and sighted subjects with eyes closed present similar lower stability in single leg stance that sighted subject with visual cues [START_REF] Giagazoglou | Static balance control and lower limb strength in blind and sighted women[END_REF][START_REF] Ozdemir | Sensorimotor posture control in the blind: superior ankle proprioceptive acuity does not compensate for vision loss[END_REF]. As consequence, the effect of compensatory mechanism using only vestibular and somatosensory systems remains difficult to assume the whole role maintaining or improving postural balance in congenitally blind subjects.

In the other way, recent studies tried to understand how congenitally blind subjects take into account auditory cues to improve their postural sway and compensate visual deprivation [START_REF] Morgan | Sensory perception: supernormal hearing in the blind?[END_REF][START_REF] Easton | Auditory cues for orientation and postural control in sighted and congenitally blind people[END_REF]. Auditory skills could be developed by congenitally blind subjects as compensation mechanism that could help them to improve their spatial orientation and mapping environment [START_REF] Jeka | Haptic cues for orientation and postural control in sighted and blind individuals[END_REF][START_REF] Easton | Auditory cues for orientation and postural control in sighted and congenitally blind people[END_REF]. Lessard et al have shown that early blind subjects can develop mapping information using auditory cues as least as accurate than sighted subjects [START_REF] Lessard | Early-blind human subjects localize sound sources better than sighted subjects[END_REF]. According to Gori et al, congenitally blind subjects rely strongly auditory cues to orient them in environment [START_REF] Gori | Impairment of auditory spatial localization in congenitally blind human subjects[END_REF]. Spatial representations of subjects have variations in accurate perception of the space using auditory cues around their body. In back space of the body, lack of visual stimuli increase noticeable brain selectivity of auditory information [START_REF] Aggius-Vella | Audio Spatial Representation Around the Body[END_REF]. In this way, subjects present better accurate space mapping when they perceive auditory cues from their back chest than when they receive auditory information auditory cues coming in front of them [START_REF] Aggius-Vella | Audio Spatial Representation Around the Body[END_REF]. Blind subjects present functional visual cortex activation of primary and secondary visual areas during auditory localization [START_REF] Gougoux | A functional neuroimaging study of sound localization: visual cortex activity predicts performance in early-blind individuals[END_REF].

To our knowledge, any study focused on congenitally blind subjects in upright single and two legs standing posture taking into account auditory cues. The aim of the present study is to define the influence of an auditory cue in back space on one and two legs balance posture in congenitally blind subjects. We hypothesis that congenitally blind subject improves his postural stability in presence of sound in back space, compared to sighted subjects with open eyes.

Material and Methods

Eleven congenitally blind subjects (10 males and 1 female) with respective mean ± standard deviation age = 22.9 ± 1.2 years, body mass = 76.4 ± 2.5 kg and height = 181.82 ± 3.43 cm participated in this study. Congenitally blind subjects had no vision from birth and were proficient in braille readers. Evaluation of severity of vision loss was based on ophthalmological examination.

Blind participants had visual acuity less than 20/400 in better eye or visual field less than 10° around central fixation [START_REF] Vashist | Definition of blindness under National Programme for Control of Blindness: Do we need to revise it?[END_REF]. Eleven sighted subjects with similar weight and height characteristics (9 males and 2 females, age = 23.3 ± 1.4 years, body mass = 74.4 ± 5.2 kg and height = 173.36 ± 6.73 cm) were also enrolled. Sighted subjects were healthy without any visual impairment. All subjects (Sighted and blind) were right handed dominant and presented any deficit in other sensory system. All subjects were undergraduate and graduate students in caregiver or manual therapy and gave informed consent prior participation. The study was approved by the local ethic committee, and complied with the Helsinki declaration.

Each subject stood on a force platform (Neuro Com Balance Master) that recorded CoP at 100 Hz sampling frequency. Subject was asked to stand in upright posture on the force plate in natural position, with arms along his body, looking forward (with a target attached forward their eyes for sighted subject). A loudspeaker placed 2 meters behind the subject at 1.2 m height was used in order to present a sound at 40 dB during each subject's upright posture. The loudspeaker's position was fixed approximately on behind the subject's chest in order to improve spatial representation around the body using auditory skill [START_REF] Aggius-Vella | Audio Spatial Representation Around the Body[END_REF]. The sound stimulus delivered by the loudspeaker was an alarm beep with principal component at 1000 Hz [START_REF] Aggius-Vella | Audio Spatial Representation Around the Body[END_REF].

During the experiment, participants were tested under four unipedal conditions and two bipedal conditions. For each condition, three trials were held, meaning a total of 18 trials (table 1). First, the subject performed 10 s trials of unipedal quiet stance [START_REF] Giagazoglou | Static balance control and lower limb strength in blind and sighted women[END_REF][START_REF] Ozdemir | Sensorimotor posture control in the blind: superior ankle proprioceptive acuity does not compensate for vision loss[END_REF] on each foot (left and right) on the force plate according to Neuro Com software recommendations. Three unipedal trials on each foot (left and right) were performed with auditory stimulus. Three other unipedal trails on each foot were performed without sound. The subject had two minutes' break between each quiet stance. Then, the subject performed six 30 s trials bipedal quiet standing with feet placed on the force plate with angle of 30 degrees between midpoint of heel and distal end of great toe and 10 cm between internal malleolus. As for unipedal trials, the subject performed three trials with auditory stimulus and three others without sound. All trials with auditory stimulus were realized with a constant auditory signal during the whole trial time. Auditory stimulus was presented in randomized order for unipedal and bipedal conditions. Neuro Com software converts subject's CoP data into mean sway velocity (deg.s -

1

) for each trial. Mean sway velocity of the three trials were computed in each condition (with and without sound in unipedal and bipedal conditions). In bipedal condition, only the 10 s at the middle time of the trial were used to compute mean sway velocity [START_REF] Chkeir | Balance quality assessment as an early indicator of physical frailty in older people[END_REF] in the aim to compare unipedal and bipedal conditions. Shapiro-wilk test confirmed the normality of the mean sway velocities in each condition. Student test were used to compare significant difference between blind and sighted subjects with sound and without sound conditions. Statistical significance was accepted at p < 0.05. 2) showed that blind subjects (B) had significant higher mean sway velocity than sighted subjects (S) in left unipedal upright posture without sound (VCoP B = 1.47 ± 0.21 deg.s -1 vs VCoP S = 0.79 ± 0.13 deg.s -1 ; p = 0.001). Blind subjects had also significant higher mean sway velocity than sighted subjects in left unipedal upright posture with auditory signal condition (VCoP B = 1.7 ± 0.46 deg.s -1 vs VCoP S = 1.08 ± 0.32 deg.s -1 ; p = 0.001).

Results

Student test results (table

Blind subjects had significant higher mean sway velocity than sighted subjects in right unipedal upright posture without sound (VCoP B = 1.88 ± 0.27 deg.s -1 vs VCoP S = 0.95 ± 0.14 deg.s -1 ; p = 0.001). Blind subjects had also significant higher mean sway velocity than sighted subjects in right unipedal upright posture with auditory signal condition (VCoP B = 1.55 ± 0.49 deg.s -1 vs VCoP S = 1.02 ± 0.02 deg.s -1 ; p = 0.001).

Blind subjects had significant lower mean sway velocity than sighted subjects in bipedal upright posture without sound (VCoP B = 0.27 ± 0.11 deg.s -1 vs VCoP S = 0.4 ± 0.14 deg.s -1 ; p = 0.02). Blind subjects had no significant difference in mean sway velocity compared to sighted subjects in bipedal upright posture in auditory signal condition (VCoP B = 0.39 ± 0.28 deg.s -1 vs VCoP S = 0.38 ± 0.15 deg.s -1 ; p = 0.88).

The Results of sighted subjects mean say velocity of the present study were consistent with previous study of sighted healthy subject with similar age, height and mass [START_REF] Hammami | Comparison of static balance and the role of vision in elite athletes[END_REF].

Discussion

To better understand how auditory cues placed behind them influence congenitally blind subjects postural balance in quiet stance, we examined the differences in unipedal and bipedal upright posture in two auditory conditions (with auditory stimulus and without sound) between a group of congenitally blind subjects and a group of healthy sighted subjects. The overall results showed that congenitally blind subjects in unipedal quiet stance didn't use auditory cues ideally placed on their bodies back to improve their balance posture. However, in bipedal quiet stance, congenitally blind subjects presented similar balance posture as open eyes sighted subjects in quiet stance associated with auditory signal placed behind them.

The results of the present study in unipedal quiet stance are in agreement with results of Ozdemir et al, realized without sound where blind subjects increased their sway amplitude on each one leg stance more than sighted subjects with eyes open [START_REF] Ozdemir | Sensorimotor posture control in the blind: superior ankle proprioceptive acuity does not compensate for vision loss[END_REF]. Although blind subjects could better involve their somatosensory systems, especially at ankle proprioception, they presented lower balance stability in one leg stance than sighted subjects [START_REF] Ozdemir | Sensorimotor posture control in the blind: superior ankle proprioceptive acuity does not compensate for vision loss[END_REF]. In the present study, auditory cue didn't improve stability in one leg stance enough to permit blind subjects to improve the balance posture as sighted subjects. As already described in other studies, the use of compensatory mechanism seems to be limited for blind subjects [START_REF] Ozdemir | Sensorimotor posture control in the blind: superior ankle proprioceptive acuity does not compensate for vision loss[END_REF][START_REF] Schmid | Equilibrium during static and dynamic tasks in blind subjects: no evidence of cross-modal plasticity[END_REF]. In the case of one leg stance, auditory cues appear to have minor effect on balance posture. In this case, blind subject seems to use larger oscillations in order to maintain posture using especially somatosensory and vestibular functions [START_REF] Ozdemir | Sensorimotor posture control in the blind: superior ankle proprioceptive acuity does not compensate for vision loss[END_REF][START_REF] Schmid | Equilibrium during static and dynamic tasks in blind subjects: no evidence of cross-modal plasticity[END_REF]. As in other daily activities (walking with gait-assistance devices, gait initiation during crosswalk, etc.) congenitally blind subjects remains essentially dependent on somatosensory functions to control balance posture one leg quiet stance [START_REF] Parreira | Postural control in blind individuals: A systematic review[END_REF]. According to Nakata et al, congenitally blind subjects may have enhanced abilities that use somatosensory system in order to maintain postural balance in specific posture in order to prevent falling risk [START_REF] Nakata | Automatic postural response systems in individuals with congenital total blindness[END_REF].

The results of the present study in bipedal quiet stance are in agreement with some previous studies realized without sound where blind subjects were characterized by lower sway amplitude than sighted subjects with eyes open [START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF][START_REF] Rougier | Adaptative effects of loss of vision on upright undisturbed stance[END_REF]. Without auditory signal, congenitally blind subjects seem to be more stable than healthy subjects. This could be explain by compensatory mechanisms where blind subjects initiate early changes in cortex activations in order to better take into account somatosensory system [START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF]. If blind subjects seem to develop less energy expenditure in order to maintain upright posture, this lower sway could limit blind subjects to unpredictable perturbations [START_REF] Rougier | Adaptative effects of loss of vision on upright undisturbed stance[END_REF]. Surprisingly, when an auditory cue was placed behind them, both blind and sighted subjects of the present study performed similar sway velocity. These results could be explained by some links between vestibular system and the expression of anxiety and emotion [START_REF] Kemoun | Thymocognitive input and postural regulation: a study on obsessive-compulsive disorder patients[END_REF]. In absence of auditory cues, congenitally blind subjects can maintain their quiet stance only using somatosensory and vestibular systems. This situation could be source of anxiety that could change the parabrachial nucleus activity which has connection with the vestibular system [START_REF] Kemoun | Thymocognitive input and postural regulation: a study on obsessive-compulsive disorder patients[END_REF]. For congenitally blind subjects, the lack of sound information limits the use of auditory cues and could disturb attention and emotional activities. These perturbations could modify blind subject skills to use his vestibular function. In this case, blind subject could only promote somatosensory system information that could limit the use of the compensatory mechanism using only joint stiffness [START_REF] Rougier | Adaptative effects of loss of vision on upright undisturbed stance[END_REF][START_REF] Maurer | Vestibular, visual, and somatosensory contributions to human control of upright stance[END_REF]. In the present study, the increase of sway amplitude in congenitally blind subjects' upright posture associated with auditory cues compared to sighted subjects could be explain as a better using of auditory, vestibular and somatosensory systems. Using auditory cues, congenitally blind subjects could better use all compensatory mechanisms in order to better maintain their balance posture and to face environment perturbations [START_REF] Schwesig | Postural control in subjects with visual impairment[END_REF][START_REF] Soares | Postural control in blind subjects[END_REF][START_REF] Easton | Auditory cues for orientation and postural control in sighted and congenitally blind people[END_REF]. These results are in agreement with the study of Weeks et al [START_REF] Weeks | A positron emission tomographic study of auditory localization in the congenitally blind[END_REF] and various other studies that have shown brain plasticity of blind subjects activating visual cortex areas associated with usual posterior parietal areas in order to map space using auditory cues [START_REF] Gougoux | A functional neuroimaging study of sound localization: visual cortex activity predicts performance in early-blind individuals[END_REF][START_REF] Striem-Amit | Visual cortex extrastriate body-selective area activation in congenitally blind people "seeing" by using sounds[END_REF].

The results of the present study could be limited by the low number of blind subjects that have participated. To our knowledge, the present study is the first that afforded more than ten congenitally blind subjects performing one leg stances [START_REF] Giagazoglou | Static balance control and lower limb strength in blind and sighted women[END_REF][START_REF] Ozdemir | Sensorimotor posture control in the blind: superior ankle proprioceptive acuity does not compensate for vision loss[END_REF]. Future studies with bigger sizes of congenitally blind subjects could be provided in order to better understand the influence of auditory cues on balance posture. Another limitation of the present study could be the absence of dominant leg in unipedal balance testing. Indeed, the present study only focuses on the influence of auditory cues on quiet stance in bipedal and unipedal posture. Due to the fact that the force plate was not able to estimate limb difference [START_REF] Promsri | How does lower limb dominance influence postural control movements during single leg stance?[END_REF][START_REF] Clifford | Postural control in healthy individuals[END_REF], complementary studies using kinematics assessment would be set up in order to investigate the influence of auditory cues on dominant one leg postural control [START_REF] Promsri | How does lower limb dominance influence postural control movements during single leg stance?[END_REF][START_REF] Clifford | Postural control in healthy individuals[END_REF].

Conclusion

The present study showed that auditory cues placed backward chest of congenitally blind subjects could increase their balance control in bipedal upright posture compared to sighted subjects. In this case, blind subjects could better use compensatory mechanisms to perform quiet standing as sighted subjects. Without sound or in unipedal upright posture, congenitally blind subjects probably have sensory perturbations or limitations that impose them adaptations in order to avoid falling risk.

Congenitally blind subjects' skills to use auditory cues can't totally compensate somatosensory and vestibular systems perturbations in postural control on one leg stance. Congenitally blind subjects seem to be more dependent to the nature of the environment and the postural task than sighted subjects. In clinical application, add auditory cues at specific points (pedestrian crossing, hospital corridor, etc.) could help congenitally blind subject in daily activities in order to prevent falling risk [START_REF] Weeks | A positron emission tomographic study of auditory localization in the congenitally blind[END_REF].

Auditory stimulus

Table 1 :

 1 Postural balance trials by condition.

	Without sound

Table 2 :

 2 Mean sway velocity (degree.m -1 ) in congenitally blind subjects compared to sighted subjects in bipodal an unipodal upright posture with auditory signal versus without sound (Mean ± standard deviations). Level of significance: † for p < 0.05 ; ‡ for p < 0.01.


Mehdi Boucheikhchoukh
email: m.boucheikhchoukh@yahoo.fr

Noureddine Mechouk
email: noureddinemechouk@gmail.com

Ahmed Benakhla
email: benakhlaahmed@gmail.com

Didier Raoult
email: didier.raoult@gmail.com

Philippe Parola
email: philippe.parola@univ-amu.fr.

Prof Parola Philippe
email: philippe.parola@univ-amu.fr

Molecular evidence of bacteria in Melophagus ovinus sheep keds and Hippobosca equina forest flies collected from sheep and horses in northeastern Algeria

Keywords: Sheep, Melophagus ovinus, Horses, Hippobosca equina, vector-borne diseases, Algeria

The sheep ked, Melophagus ovinus, and the forest fly, Hippobosca equina, are parasitic dipteran insects of veterinary importance. As hematophagous insects, they might be considered as potential vectors of diseases which may be transmissible to humans and animals. The purpose of this study was to present initial primary data about these two species in Algeria. To do so, we conducted a molecular survey to detect the presence of bacterial DNA in flies collected in Algeria. A total of 712 flies including, 683 Melophagus ovinus and 29 Hippobosca equina were collected from two regions in northeastern Algeria. Monitoring the monthly kinetics of M. ovinus infestations showed something resembling annual activity, with a high prevalence in January (21.67%) and May (20.94%).

Real-time quantitative PCR assays showed that for 311 tested flies, 126 were positive for the Bartonella spp. rRNA intergenic spacer gene and 77 were positive for Anaplasmataceae. A random selection of positive samples was submitted for sequencing. The DNA of Bartonella chomelii and Bartonella melophagi were amplified in, respectively, five and four H. equina. 25 M. ovinus positive samples were infected by Bartonella melophagi. Amplification and sequencing of the Anaplasma spp. 23S rRNA gene revealed that both species were infected by Wolbachia sp. which had previously been detected in Cimex lectularius bed bugs.

Overall, this study expanded knowledge about bacteria present in parasitic flies of domestic animals in Algeria.

Introduction

Hippoboscidae flies, usually known as keds or louse flies, are obligate hematophagous Diptera, which bite birds and some mammals [START_REF] Small | A review of Melophagus ovinus (L.), the sheep ked[END_REF][START_REF] Bequaert | The Hippoboscidae or louse-flies (Diptera) of mammals and birds. Part I. Structure, physiology and natural history[END_REF]. They are organised into more than 19 genera and 150 cosmopolitan species [START_REF] Duvallet | Entomologie médicale et vétérinaire[END_REF][START_REF] Graciolli | [END_REF][START_REF] Iwasa | Contribution to the knowledge of the Hippoboscidae (Diptera) from the Republic of Korea[END_REF][START_REF] Dick | Checklist of world Hippoboscidae (Diptera: Hippoboscoidea)[END_REF]. Members of this family, particularly those relating to sheep (Melophagus ovinus), horses (Hippobosca equina) and dogs (Hippobosca longipennis) bite their hosts and people who take care of these animals [START_REF] Small | A review of Melophagus ovinus (L.), the sheep ked[END_REF][START_REF] Duvallet | Entomologie médicale et vétérinaire[END_REF][START_REF] Zhang | Ultrastructural investigation of antennae in three cutaneous myiasis flies: Melophagus ovinus, Hippobosca equina, and Hippobosca longipennis (Diptera: Hippoboscidae)[END_REF]. Human reactions to these bites vary widely, ranging from simple redness followed by pruritic inflammation to anaphylactic shock requiring emergency treatment [START_REF] Matito | IgEmediated anaphylaxis to Hippobosca equina in a patient with systemic mastocytosis[END_REF][START_REF] Decastello | Anaphylactic reaction caused by a horse-fly species (Hippobosca equina)][END_REF][START_REF] Vidal | Anaphylaxis to Hippobosca equina (louse fly)[END_REF][START_REF] Maggi | Isolation of Candidatus Bartonella melophagi from human blood[END_REF]. In animals, these ectoparasites are responsible for weight loss, decrease in wool growth and livestock milk production, and cutaneous myiasis causing, in the long run, significant economic losses [1, 7,

12-14].

The pathogenic role of Hippoboscidae flies remains insufficiently documented. As they are often subservient to the hosts they parasitize, this could cast doubt on their ability to transmit pathogens to other animals or humans [START_REF] Duvallet | Entomologie médicale et vétérinaire[END_REF]. However, recent studies have reported the molecular detection of several vector-borne pathogens in Hippoboscidae flies collected on ruminants [START_REF] Halos | Role of Hippoboscidae flies as potential vectors of Bartonella spp. infecting wild and domestic ruminants[END_REF][START_REF] Hornok | First molecular evidence of Anaplasma ovis and Rickettsia spp. in keds (Diptera: Hippoboscidae) of sheep and wild ruminants[END_REF][START_REF] Szewczyk | Molecular detection of Bartonella spp. in deer ked (Lipoptena cervi) in Poland[END_REF][START_REF] Zhao | First report of Anaplasma ovis in pupal and adult Melophagus ovinus (sheep ked) collected in South Xinjiang, China[END_REF][START_REF] Buss | Detection of Lyme disease and anaplasmosis pathogens via PCR in Pennsylvania deer ked[END_REF][START_REF] Liu | First report of Rickettsia raoultii and R. slovaca in Melophagus ovinus, the sheep ked[END_REF][START_REF] Rudolf | Molecular survey of arthropod-borne pathogens in sheep keds (Melophagus ovinus), Central Europe[END_REF][START_REF] Martinkovic | Trypanosoma (Megatrypanum) melophagium in the sheep ked Melophagus ovinus from organic farms in Croatia: phylogenetic inferences support restriction to sheep and sheep keds and close relationship with trypanosomes from other ruminant species[END_REF], horses [START_REF] Halos | Role of Hippoboscidae flies as potential vectors of Bartonella spp. infecting wild and domestic ruminants[END_REF][START_REF] Mediannikov | Bartonellae in animals and vectors in New Caledonia[END_REF], dogs [START_REF] Rani | Hippobosca longipennis-a potential intermediate host of a species of Acanthocheilonema in dogs in northern India[END_REF][START_REF] Baker | A review of the role played by the Hippoboscidae (Diptera) as vectors of endoparasites[END_REF] and raptors [START_REF] Farajollahi | Detection of West Nile virus RNA from the louse fly Icosta americana (Diptera: Hippoboscidae)[END_REF].. All these studies support the hypothesis that Hippoboscidae flies might be vectors of infectious diseases. However, molecular studies are not sufficient to confirm the vector competence of an arthropod.

Although M. ovinus is the most studied Hippoboscidae alongside H. equina [1, 27, 28], data on these two species in terms of their biology and involvement in the transmission of pathogens are entirely lacking in Algeria. The main reason for this is that M. ovinus and H. equina flies often go unnoticed and are considered by farmers to be harmless pests.

It was from this perspective that we investigated Hippoboscidae flies that parasitize sheep and horses in northeastern Algeria. The aims of the present study were to gather initial data on the monthly prevalence of M. ovinus and high-risk periods, and to use molecular procedures to investigate the presence of bacteria in M. ovinus sheep keds and H. equina forest flies.

Materials and Methods

Study areas and period of collection

The study was carried out between August 2015 and July 2016 in two regions of the extreme north east of Algeria: El Tarf (36°51'21.5"N, 8°19'34.5"E) and Mila (36°27′0″N, 6°16′0″E) (Fig. 1). These regions are known for cattle breeding and have fairly similar climates. El Tarf is made up of two clearly differentiated areas: the northern part is mainly characterised by alluvial plains and the climate is sub-humid to warm humid; while the southern part has greater relief and the climate is humid to cool and wet [START_REF] Côte | Les étages bioclimatiques des régions de l'Est algérien[END_REF]. The Mila region is notable for its humid climate in the north, sub-humid to semi-arid in the centre, and semi-arid in the south

[29].

Fly collection and identification

Melophagus ovinus flies were collected on a monthly basis from three sheep farms located in the town of Tessala Lematai in the Mila region. These farms culminate at approximately the same altitude (~ 1,465m) and are each composed of 30 sheep of different sex and age categories. For every ovine which was examined, its age (young: ≤ 1 year; adult: >1 year) and sex were recorded. To collect the flies, the fleeces of the parasitized sheep were parted and the flies were collected directly from the deepest parts of the wool. During the shearing period in May, the flies were recovered directly and individually from the wool bales placed beside each sheared sheep. In both cases, sheep keds were carefully removed and immediately placed in 70% ethanol. The M. ovinus pupae were not recovered from infested animals and were left in situ.

The Hippobosca equina forest flies were collected only once, in June, from two horse barns in Ain El Kerma, a town in the El Tarf region. The first barn consisted of 11 horses while the second was composed of nine horses. The flies were caught manually from the inner thigh and around the perineum of parasitized horses and were directly stored in 70% ethanol.

The sampled flies were morphologically identified using a Leica® binocular lens with an LED light at the IHU Méditerranée Infection, Marseille, France. Identification was essentially based on the morphotaxonomic criteria reported in Huston and Wall and Shearer dichotomous keys [START_REF] Hutson | Keds, Flat-flies and Bat-flies. Diptera, Hippoboscidae and Nycteribiidae. Handbooks for the Identification of British Insects[END_REF][START_REF] Wall | Veterinary ectoparasites: biology, pathology and control[END_REF]. Photographs of the dorsal and ventral sides of each species were taken with a microscope at a magnification of ×56 (Zeiss Axio Zoom.V16, Zeiss, Marly le Roi, France) (Fig. 2 and3).

DNA extraction

DNA extraction was performed on a representative selection of M. ovinus flies from each of the three sheep farms and on all the H. equina fly specimens. All experiments and sample handling was conducted under sterile conditions under a laminar flow biosafety hood. The sample preparation process was the same for both fly species. The flies were removed from the ethanol, rinsed for 10 minutes in a sterile distilled water bath and then dried with filter paper. For each sample, a longitudinal incision was made using a scalpel blade, cutting the fly into two equal parts. One half was dropped into a sterile tube (Eppendorf; Hamburg, Germany) while the remaining part was kept at -20°C for further analysis.

Each half-fly was then incubated at 56°C overnight with 180µL of G2 lysis buffer (Qiagen, Hilden, Germany) and 20µL proteinase K (Qiagen, Hilden, Germany). DNA extraction was processed using an EZ1 ® DNA Tissue Kit (Qiagen, Hilden, Germany) and EZ1 ® BioRobot ® extraction device according to the manufacturer's recommendations. Between each batch, all parts of the device were disinfected and subjected to 20 minutes of ultraviolet light to avoid any cross contamination. Finally, the DNA from each sample was eluted in 100 µL of Tris-EDTA (TE) buffer (Qiagen, Hilden, Germany) and stored at -20°C under sterile conditions.

Molecular survey and PCR amplification

To investigate the presence of bacterial DNA, all the extracted DNA was subjected to realtime PCR assay using a CFX ConnectTM Real-Time PCR Detection System (Bio-Rad, Marne la Coquette, France) targeting a fragment of specific genes of five bacteria. The citrate synthase (gltA) "RKND03" gene was used to detect Rickettsia spp. The PCR products were purified using a Macherey-Nagel plate (NucleoFast® 96 PCR, Düren, Germany), as recommended by the manufacturer. Amplicons sequences were obtained using the ABI Prism 3130xl (ABI PRISM, PE Applied Biosystems, USA) genetic analyser capillary sequencer and the BigDye® Terminator v1.1, v3.1. 5x Sequencing Buffer (Applied Biosystems, Warrington, United Kingdom).

Sequence processing

In order to identify bacterial species, the obtained nucleotide sequences were first assembled and edited using ChromasPro v.1.7.7 software (Technelyium Pty. Ltd., Tewantin, Queensland, Australia). They were then processed by comparing them with the sequences available in the GenBank database, using the online Basic Local Alignment Search Tool (BLAST) (http:// blast.ncbi.nlm.nih.gov/Blast.cgi).

Statistical analysis

Statistical analyses were performed using the SPSS v24.0 HF02 software (IBM SPSS Statistics for Windows, Version 24.0, Armonk, NY: IBM Corp, 2016), and the Pearson's chisquared "χ2" test was used to compare the overall prevalence of M. ovinus according to the month of collection and the age and sex of the sheep.

Ethical considerations

Ethical consent for sampling from sheep and horses was granted by the El Tarf University Animal Ethics Committee. Verbal agreement for the field study was obtained from the animals' owners and from the local agricultural services office.

Results

Fly collection and infestation prevalence

A total of 683 adult M. ovinus flies (320 males and 363 females) and 29 H. equina flies (14 males and 15 females) were collected respectively from 81/90 (90%) infested sheep belonging to the three Mila farms and 9/20 (45%) infested horses from the El Tarf barns.

The sheep studied here were also co-infested by other ectoparasites such as Damalinia ovis lice, and Haemaphysalis sulcata and Haemaphysalis punctata ticks, while the horses were infested by Damalinia equi lice and Boophilus (Rhipicephalus) annulatus ticks.

Monitoring of the monthly kinetics of M. ovinus infestation in the three sheep farms showed that these flies parasitize animals almost all year round, with a high prevalence in January (21.67%) and May (20.94%) and almost zero prevalence in autumn and summer (Fig. 4).

These results allow us to define a statistically significant (χ2 test, ρ = 0.01) period of activity for this fly species which can be estimated to exist between the months of October and May.

In addition, we compared the ovine infestation rate by the age and sex of the sheep. The results revealed that there was no statistically significant difference in M. ovinus infestation rates between male and female sheep (χ2 test, ρ = 0.58) and between young and adult sheep (χ2 test, ρ = 0.21).

The horses' infestation by the H. equina forest fly could not be correctly monitored due to the frequent sales of the horses, which resulted in off-peak periods where no horses were present in the stables.

Detection of bacteria

From 712 collected flies, 311/712 specimens including, 282 M. ovinus (94 specimens from each of the three farms) and 29 H. equina were randomly selected and screened using realtime PCR for the presence of bacteria. Of the 311 flies tested, 202/311 (64.95%) samples were positive for at least one of the investigated bacteria, while 58/311 (18.64%) were found to be co-infected by two bacterial genus. The 311 flies tested negative for all spotted fever group Rickettsia species, Borrelia spp., and C. burnetii. The bacterial identity of a random selection of the positive samples was later achieved by standard PCR amplification and sequencing.

Real-time assays targeting the intergenic spacer (ITS) gene for Bartonella spp. revealed that 126/311 (40.51%) flies, including 104/282 (36.87%) M. ovinus and 22/29 (75.86%) H. equina collected respectively from sheep and horses, were Bartonella-positive. From 126 Bartonellapositive samples, all 22/29 H. equina specimens and 25/104 randomly selected M. ovinus specimens were subject to standard PCR. However, amplification trials using the ITS gene were not successful. Sequencing analysis using the gltA system was subsequently performed and high quality sequences were obtained for all (25/25) M. ovinus specimens and 9/22 of the H. equina flies. The BLAST analysis results revealed 99.48 to 100% identity with the corresponding 753 base pair fragment of Bartonella chomelii (GenBank accession nos. KM215691.1 and KM215693.1) for 5/9 sequences amplified from H. equina and 100% identity with Bartonella melophagi (GenBank accession nos. MG701237.1) for the remaining sequences (4/9). However, following the BLAST query, all sequences obtained from M. ovinus specimens showed 99.50 to 100% identity with the corresponding 323 base pair fragment of B. melophagi (GenBank accession nos. MG701237.1).

Of the total 23S qPCR screened flies, 77/311 samples (24.75%) involving 73/282 M. ovinus (25.88%) and 4/29 H. equina (13.79%) were infected by bacteria from the Anaplasmataceae family. Within this, 23S rRNA amplification and sequencing was successful for 65/77 samples (61 M. ovinus and four H. equina). The query of the resulting sequences against the NCBI GenBank database showed, for both fly species, 99.31 to 100% identity with the "Wolbachia endosymbiont of Cimex lectularius" sequence (GenBank accession nos. AP013028.1).

Discussion

Over last decade, the vast majority of research conducted on parasitic arthropods of animals in Algeria has essentially focused on ticks, fleas, mosquitoes, and sand-flies. The interest in these categories of pests largely reflects the fact that they can significantly affect their hosts by transporting and inoculating several vector-borne diseases

Conclusion

In this study, we provided molecular evidence for the presence of bacteria in two fly species collected from sheep and horses in northeastern Algeria. B. chomelii has been detected for the first time in Algeria and the African continent, while B. melophagi is reported for the first time in Algeria. In view of these facts, more attention should be given to livestock haematophagous flies, since they can carry zoonotic pathogens. Measures to combat them could be conducted according to high-risk periods. However, the choice of the most effective insecticide will have to be made according to two parameters: its long persistence and its quality/price ratio.

[ 32 ]

 32 , the intergenic spacer (ITS2) gene was used to detect Bartonella spp.[START_REF] Mediannikov | Looking in ticks for human bacterial pathogens[END_REF], the 23S ribosomal RNA gene was used to detect the Anaplasmataceae bacteria [34], the ITS4 spacer was used to detect Borrelia spp.[35], and IS30A spacers were used to detect Coxiella burnetii[START_REF] Mediannikov | Coxiella burnetii in humans and ticks in rural Senegal[END_REF].15µl of the qPCR reaction mix, without any DNA, and dilutions of DNA extracts of cultured bacteria strains were used in each test respectively as negative and positive controls, as previously described[START_REF] Aouadi | Molecular evidence of tick-borne hemoprotozoan-parasites (Theileria ovis and Babesia ovis) and bacteria in ticks and blood from small ruminants in Northern Algeria[END_REF]. Samples were considered positive when the cycle threshold (Ct) value given by the Bio-Rad CFX Manager™ v3.1 software (Bio-Rad, Marne la Coquette, France) was ≤35. A random selection of these positive samples was subsequently subject to conventional PCR prior to sequencing in order to identify pathogens at the species level.Conventional PCR analysis was performed using an automated DNA thermal cycler (Applied Biosystems, 2720, Foster city, USA), targeting the intergenic transcribed (ITS) gene[START_REF] Kernif | Molecular Evidence of Bartonella Infection in Domestic Dogs from Algeria, North Africa, by Polymerase Chain Reaction (PCR)[END_REF] and the citrate synthase (gltA) gene[START_REF] Dahmani | Bartonella bovis and Candidatus Bartonella davousti in cattle from Senegal[END_REF] for qPCR Bartonella spp. positive samples. Bacterial DNA from the Anaplasmataceae family was detected using the 23S gene[START_REF] Dahmani | Molecular detection of Anaplasma platys and Ehrlichia canis in dogs from Kabylie, Algeria[END_REF]. Amplified products were then subjected to electrophoresis through a 0.5% agarose gel stained with SYBR Safe™ and viewed using a ChemiDoc™ MP ultraviolet imager (Bio-Rad, Marnes-la-Coquette, France).

  [START_REF] Aouadi | Molecular evidence of tick-borne hemoprotozoan-parasites (Theileria ovis and Babesia ovis) and bacteria in ticks and blood from small ruminants in Northern Algeria[END_REF][START_REF] Lafri | Detection of relapsing fever Borrelia spp., Bartonella spp. and Anaplasmataceae bacteria in argasid ticks in Algeria[END_REF][START_REF] Bessas | Molecular evidence of vector-borne pathogens in dogs and cats and their ectoparasites in Algiers, Algeria[END_REF][START_REF] Bitam | Molecular detection of Rickettsia typhi and Rickettsia felis in fleas from Algeria[END_REF][START_REF] Boubidi | Plasmodium falciparum malaria, southern Algeria[END_REF][START_REF] Berdjane-Brouk | First detection of Leishmania infantum DNA in Phlebotomus longicuspis Nitzulescu, 1930 from visceral leishmaniasis endemic focus in Algeria[END_REF], which can in turn lead to considerable economic losses.To date, few entomological investigations into haematophagous flies and their associated microorganisms have been the subject of research in Algeria. The only available records were inventories on Nycteribiidae bat flies, which may be recognized as potential vectors of Bartonella tamiae[START_REF] Leulmi | Detection of Bartonella tamiae, Coxiella burnetii and rickettsiae in arthropods and tissues from wild and domestic animals in northeastern Algeria[END_REF][START_REF] Bendjeddou | Bat ectoparasites (Nycteribiidae, Streblidae, Siphonaptera, Heteroptera, Mesostigmata, Argasidae, and Ixodidae) from Algeria[END_REF]. The unpredictable behaviour of the Hippoboscidae fly its voracity and its spectacular ability to reproduce and quickly increase the number of individuals per population, as well as and its ability to cover long distances in flight, has significantly increased the vulnerability of animals to vector-borne pathogens. Consequently, the flybacteria relationship should not be overlooked.In this study, we have observed the prevalence and the period of occurrence of the M. ovinus sheep ked by conducting an annual monitoring of the infestation. An overall prevalence of 90% for M. ovinus registered in our study can be explained by the combination of various crucial factors including the presence of a cold climate and a high altitude region which are absolutely essential to the fulfilment of M. ovinus life cycle [1, 48]; The mismanagement of the livestock and unhealthy husbandry conditions which could predispose malnourished sheep to infestation [49]; and finally the highly abusive use of insecticides which could lead to the emergence of a resistant cohort of keds [12]. In addition, the annual monitoring of M. ovinus population revealed that the number of sheep keds began to increase in late October, peaked in January and May and observed a seasonal low starting from late May. These findings are in compliance with previous observations where M. ovinus populations are reported to be active in winter and spring and less in summer [50, 51]. On the other side, the lack of a statistically significant difference in infestation rates of M. ovinus depending on the sex and ages of infested sheep is consistent with previous studies where the abundance of M. ovinus flies in sheep was shown to be related to abiotic components rather than host dependent factors [1, 49]. Molecular tools were used to conduct the bacteriological survey. The reliability of these techniques in such epidemiological investigations on arthropods and especially on flies has been demonstrated in previous research [15, 16, 20, 49]. Furthermore, the results reported here were obtained under sterile conditions using meticulous laboratory procedures and sophisticated instruments routinely employed in our laboratory. Although a negative and positive control of each PCR test was used to confirm the accuracy of our results, this cannot confirm the vector role of the flies studied, as they could have acquired the bacteria after blood meals on infected animals or crossed other vectors and co-fed with them. This survey demonstrated that 64.95% of tested flies were infected by at least one bacteria. B. melophagi was found in M. ovinus and H. equina while B. chomelii was detected in H. equina. Phylogenetically, these two bacteria are affiliated, alongside B. bovis, B. capreoli and B. schoenbuchensis, to Bartonella strains which mainly infect ruminants [52]. However, their pathogenicity and the way that they may be transmitted are poorly understood. Nevertheless, the manifestation of symptoms in patients who are in contact with animals and who suffered from idiopathic pericarditis, circular red skin lesions, and chronic asthenia have been associated with the clinical expression of a B. melophagi infection which was later isolated from their blood [11]. Although sheep were reported as the most likely reservoirs of B. melophagi [11, 53, 54] there is no symptomatic description of such infections in animals. further investigation of B. melophagi are needed. Bartonella chomelii has not yet been shown to be pathogenic [57-59]. This Bartonella species was first isolated from domestic cattle blood [60] and was later identified as the most common Bartonella infecting cattle [61]. Both species of Bartonella adapted to mammal reservoirs and, according to previous studies, they share biting flies as common potential vectors [54]. This symbiosis is possible when a variety of adaptation, metabolic interconnections and genomic changes occur between insects and bacteria. The occurrence of B. melophagi within M. ovinus sheep keds has already been reported in previous works [21, 49, 54, 62, 63] and B. melophagi DNA was found in the gut of M. ovinus adults and even in the pupal stages, thus suggesting this bacteria is endosymbiotic in nature, and confirming our findings [15, 21, 49, 62, 64, 65]. However, B. melophagi had never been detected in H. equina. To the best of our knowledge, this is the first report of such an association. The few available reports until now have associated H. equina flies with B. schoenbuchensis and B. chomelii [15, 23]. Bartonella chomelii has been associated with several arthropods, such as Ixodid ticks and biting flies [15, 66, 67]. However, its most effective vector remains unknown. Overall, the flies sampled in this survey were infected by two Bartonella species and, as alluded to above, this does not guarantee their role as vectors. Further investigations are needed to shed light on the role of H. equina in the epidemiology of B. melophagi and B. chomelii. This study also investigated the presence of bacteria from the Anaplasmataceae family. The ability of hippoboscidae flies to vector bacteria from this family has been recently assessed. The results confirmed the presence of Anaplasma ovis in M. ovinus specimens and its vertical transmission among keds [16, 18], thus bringing new evidence regarding the potential position of these flies as mechanical or biological vectors. In our case, "Wolbachia endosymbiont of Cimex lectularius" was detected in 61 M. ovinus and four H. equina. To the best of our knowledge, this study provides the first molecular proof of the presence of Wolbachia spp. DNA in H. equina adults. However, these bacteria have already been described as the third most common bacteria genus after Bartonella and Arsenophonus in the midgut of M. ovinus [63, 64]. In addition, the screening of M. ovinus microbiota revealed that Wolbachia was present intracellularly in various tissues such as adipocytes, secretory cells and intestinal tissue [65]. These multiple locations of Wolbachia spp. have previously been described for other arthropods such as lice and bed bugs [68]. However, no studies have so far reported the presence of Wolbachia spp. or Anaplasma spp. in H. equina flies.

Fig. 1 :

 1 Fig. 1: Collection sites of Melophagus ovinus and Hippobosca equina and their infection rates

Fig. 2 :

 2 Fig. 2: The "false sheep louse" Melophagus ovinus (Diptera: Hippoboscidae): A 4-7 mm fly

Fig. 3 :Fig. 4 :

 34 Fig. 3:The "forest fly" Hippobosca equina (Diptera: Hippoboscidae), 7-8 mm, horse, cattle

Acknowledgments

This work was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection and the "Investissements d'avenir" programme managed by the French National Research Agency (Méditerranée Infection 10-IAHU-03).

Competing interests

The authors declared that they have no competing interests.

Author contributions

MB contributed to arthropod collection, performed DNA extractions, PCRs, sequencing and prepared the first draft of the paper. NM helped with fly sampling and contributed to the manuscript. AB contributed to conceiving, designing and coordinating the study. DR contributed reagents/materials/analysis tools. PP * coordinated experiments and reviewed the paper.