Mahdi Khoramshahi
email: khoramshahi@isir.upmc.fr

Alexis Poignant

Guillaume Morel

Nathanael Jarrassé

A Practical Control Approach for Safe Collaborative Supernumerary Robotic Arms

Supernumerary robotic arms have a high potential to increase human capacities to perform complicated tasks; e.g., having a third arm could increase the user's strength, precision, reachability, and versatility. However, having a robotic manipulator working in extreme proximity to the user raises new challenges in terms of safety; i.e., uncontrolled and hazardous collisions with the user's body parts and the environment. In this preliminary work, we show that most of these safety considerations can be extracted from standardized norms and translated into kinematics constraints for the robot. Thus, we propose a quadratic programming approach to achieve safe inverse-kinematics and physical interaction for supernumerary arms. We validate our approach in designing a safe supernumerary arm using the 7-Dof Kinova ® Gen3 robot.

I. INTRODUCTION

The Supernumerary Robotic Limbs (SRL) are wearable manipulators with the aim to increase humans' capacities to perform complex tasks through sensorimotor augmentation; e.g., to improve human strength, precision, versatility [START_REF] Gopinath | A generative physical model approach for enhancing the stroke palette for robotic drummers[END_REF], grasping capacity [START_REF] Hussain | Using the robotic sixth finger and vibrotactile feedback for grasp compensation in chronic stroke patients[END_REF], and perception [START_REF] Wang | Building multi-modal sensory feedback pathways for srl with the aim of sensory enhancement via bci[END_REF]. Such extra artificial degrees of freedom can be added to the human body in several fashions: supernumerary arms [START_REF] Parietti | Supernumerary robotic limbs for aircraft fuselage assembly: body stabilization and guidance by bracing[END_REF], legs [START_REF] Parietti | Design and control of supernumerary robotic limbs for balance augmentation[END_REF], fingers [START_REF] Hussain | A soft supernumerary robotic finger and mobile arm support for grasping compensation and hemiparetic upper limb rehabilitation[END_REF], tails [START_REF] Xie | Augmenting human with a tail[END_REF], etc. These physical devices are intended to be lightweight with the possibility to be interfaced with human motor control; for example through brain-machine interfaces [START_REF] Eden | Principles of human movement augmentation and the challenges in making it a reality[END_REF]. The first generation of SRLs were mostly weight-supporting devices, but the field progressed toward versatile, autonomous, and assistive SRLs. Currently, SRLs are applicable to industrial settings such as assembly lines [START_REF] Parietti | Supernumerary robotic limbs for aircraft fuselage assembly: body stabilization and guidance by bracing[END_REF], health-care where SRLs provide assistance for people with disabilities [START_REF] Masia | Soft wearable assistive robotics: exosuits and supernumerary limbs[END_REF], surgical robotics [START_REF] Abdi | Control of a supernumerary robotic hand by foot: An experimental study in virtual reality[END_REF], and many other domains; see [START_REF] Prattichizzo | Human augmentation by wearable supernumerary robotic limbs: review and perspectives[END_REF] for a recent review.

In this work, we are concerned with redundant supernumerary arms with the goal to improve the manipulability -in its broad sense-of the human user. However, having such "third arms" poses new challenges when a powerful device operates in extreme proximity to the human-user. The risk of physical impacts that can lead to injuries raises new safety issues for SRLs. Most often, these issues are addressed from a mechatronic point of view [START_REF] Bright | Supernumerary robotic limbs for human augmentation in overhead assembly tasks[END_REF]; e.g., by considering backdrivable motor, serials elastic actuators, advance sensing of contact, emergency stops for the actuators, etc. There is also a growing interest in utilizing intrinsically safe structures such as soft materials for wearable robots [START_REF] Walsh | Human-in-the-loop development of soft wearable robots[END_REF], [START_REF] Nguyen | Fabric soft poly-limbs for physical assistance of daily living tasks[END_REF]. However, relying fully on mechatronics and soft materials Fig. 1. Our robotic setup where the the Kinova ® robot is worn using a vest. In this current setup, the power and the controller are off-board. can severely limit the strengths and efficacy of SRLs. On the other hand, the safety issues of SRLs are less addressed from the control point of view [START_REF] Prattichizzo | Human augmentation by wearable supernumerary robotic limbs: review and perspectives[END_REF] while there is a lack for a safe control framework in this literature; for instance, solving the Inverse Kinematics (IK) in a safe manner to avoid hazardous collision with the user, or destabilizing the user stability.

In this work, we consider a case in which the human-user provides input commands for the position of the end-effector (3 DoF) of a typical 7-DoF redundant robotic manipulator which leaves 4 DoF to satisfy the safety measures. In this manner, we reach an effective balance between the robot and human autonomy. To answer safety issues, we propose to use Quadratic Programming (QP) to solve the IK where the safety considerations can be embedded as inequality constraints for the robot joint velocities. QP is commonly used in the literature to safely generate impact in the environment [START_REF] Wang | Impactaware task-space quadratic-programming control[END_REF], collision-free path planning for robotic applications [START_REF] Wang | Collision-free navigation of autonomous vehicles using convex quadratic programmingbased model predictive control[END_REF], full-body IK of a humanoid robot with high DoF [START_REF] Feng | Optimization based full body control for the atlas robot[END_REF] or as a fast way to solve IK (to avoid computing pseudo inverse) [START_REF] Escande | Fast resolution of hierarchized inverse kinematics with inequality constraints[END_REF]. Moreover, the QP approach is suitable for SRLs which require reactive motion planning; i.e., the robot needs to continuously react to human inputs and the interaction forces with the environment. Furthermore, we consider an admittance control loop to safely react to interaction forces [START_REF] Duchaine | Safe, stable and intuitive control for physical human-robot interaction[END_REF], [START_REF] Keemink | Admittance control for physical human-robot interaction[END_REF]. Nevertheless, obtaining quantified safety measures for SRLs is a challenge as well since there is no standardized or conventional approach in the literature. In this work, we propose to use the standardized ISO norms to extract inequality constraints for the QP solver. This enables our control architecture to provide a safe behavior for a supernumerary arm; i.e., to respect safety constraints on the workspace, the velocities, the interaction forces, and the impacts. To validate our approach, we use a commercial collaborative robot (7 DoF Kinova ® Gen3 arm) is mounted on the back of the user.

II. METHOD A. Robotic setup: hardware

The Kinova ® Gen 3 Ultra lightweight robot L53 0007 is a CE-marked arm that is designed according to major safety standards. It fulfills several directives and standards such as Machinery Directive 2006/42/EC and ISO 12100:2010. Its power adapter cable is fitted with an integrated emergency stop (E-stop) button which is located near the experimenter and can be used to shutdown the robot. In case of a shutdown, the robot falls slowly due to the regenerative brakes. An additional emergency stop is also placed serially on an extension of the power cable and is placed close to the robot operator. As illustrated in Fig. 1 the robot is mounted on the back of the user. Similar setups can be found in the literature [START_REF] Bright | Supernumerary robotic limbs for human augmentation in overhead assembly tasks[END_REF], [START_REF] Ciullo | A supernumerary soft robotic hand-arm system for improving worker ergonomics[END_REF]. Kinova Gen 3 is suitable for cobotics applications given its intrinsic mechanical properties [START_REF] Kinova | User guide: Kinova gen 3[END_REF]. Additionally, the native robot control library applies a number of different joint limits for safety purposes. This includes limits on joint position, speed, acceleration and torques. Gen3 also provides a low-level admittance control mode allowing a safe physical interaction with the environment. The native control library also monitors the feedback of the internal sensors of the robotic arm, along with the heat of internal components, and in case of faulty behavior, switches the robot to a safe compliant state. For the sensory information, we rely on the conventional robotic states; i.e., joint position, velocity, and torques. Beside a Robotiq two-finger gripper (2F-85) mounted on the end-effector, we do not use any other equipment such as sensors or tracking systems.

B. Control architecture

At the lowest level, a joint-velocity controller is provided by Kinova. To simplify the high-level control, we use a 3D joystick to command a desired velocity for the end-effector. To ensure a safe robotic execution of the human-commanded task, our mid-level controller needs to satisfy several constraints which are extracted from standardized norms. For instance, while moving the end-effector based on human commands, the robot needs to stay in a safe/designated workspace and avoid high-force exertion or high-power impacts. To this end, we propose a safe controller based on Quadratic programming. In this fashion, we solve the IK problem (converting human-user commands into proper joint velocities) while respecting different safety specifications. Given general safety specifications for pHRI and our specific scenarios, we compute a set of limits on different aspects of the robotic performance; i.e., workspace, joint positions, joint velocities, interaction forces, and mechanical power. These limits, along with the current robot's state, are used to compute a set of constraints for the QP-solver. This process is repeated in each time-step given the updated measurements and user's input which leads to a reactive behavior. Our midlevel controller is implemented in [START_REF]Robotic operating system[END_REF] which runs at 40 Hz. In case of a hard collision, the control is passed to the lowlevel admittance controller of the robot; i.e., the task and other ROS-dependent components will not affect the robot's behavior until the issue is resolved by the human-user. The QP problem is solved sequentially when the solution of each time-step serves as the warm-start for the next one. For this purpose, we use qpOASES library [START_REF] Ferreau | qpoases: A parametric active-set algorithm for quadratic programming[END_REF]. Some of the main technical parameters are set as follows: terminal tolerance 1e -5, bound tolerance 1e -5, max iteration 1e3.

C. Constrained inverse kinematics using QP

We use a QP-based approach inspired by [START_REF] Suleiman | On inverse kinematics with inequality constraints: new insights into minimum jerk trajectory generation[END_REF] to solve the following IK problem:

min u,δ u T W u + δ T Qδ + g T u s.t. Ju + δ = ẋd u min < u < u max δ min < δ < δ max b min < Au < b max (1)
where the decision variables u ∈ R n represents the joint velocity commands. W is a diagonal and positive semidefinite matrix representing the corresponding costs for moving each joint. We use W = diag([0.1, 1, 10, 10, 1, 1, 1]) which is chosen experimentally with high values for the 3rd and 4th joints as they affect the posture more than other joints. In the second term, δ ∈ R m represents the slack variables with its associated weight matrix Q ∈ R n×n ≻ 0 which is defined in a manner that ||Q|| >> ||W ||. For our implementation, we choose Q = 1e8I n where I n is the identity matrix of size n. We use the linear part (g ∈ R n ) to favor an upright posture (q 2 = 0) in the null-space by choosing g = [0, k n q 2 , 0, 0, 0, 0, 0] T where q 2 is the position of the second joint, and k n is the null-space stiffness. The goal of QP is to follow the desired end-effector velocity ( ẋd ∈ R m ) while respecting the safety constraints. The equality constraint represents the forward kinematics of the robot where J ∈ R m×n is the Jacobian matrix. It can be shown that neglecting the inequality constraints and the slack variables leads to the standard IK solution as:

u = J # ẋd + (I -J # J) qn (2)
where J # = W -1 J T (JW -1 J T ) is the weighted Moore-Penrose pseudo-inverse and qn = -g/2 is the null-space velocity. In our control architecture, the desired velocity is computed as follows:

ẋd = ẋt + ẋa (3)
Where the task-related velocities ẋt ∈ R m are received from a high-level controller; e.g., a simple control interface using joystick or gaze. The second term ẋa ∈ R m is generated by the admittance loop which handles the interaction forces with respect to soft and hard collisions.

D. Safety constraints for QP

In this section, we present how the safety measures for the robot are translated into the boundaries for the inequality constraints in the QP problem.

Velocity safety: ISO 10218-1:2011 [START_REF]&2:2011, Safety requirements for industrial robots[END_REF] specifies that the maximum velocity of the robot end-effector should be 0.25m/s, to leave enough time to the operator to avoid or stop the robot. Therefore, we limit the commanded velocities for the EE at 0.2m/s. Furthermore, we limit the joint velocities at qmax = 0.3rad/s to have reasonable linear velocities for other robotic parts. This is a conservative value that is reached experimentally. The QP inequality constraints for our decision variable constraints the joints velocities as follows:

-qmax ≤ u ≤ qmax (4)
Angular position safeties: In order to take into account the mechanical limits of the robot, especially joint limits, we need to add angular position limits. However, as our command work is a velocity command, we need to express joint limits as velocity limits. To do so, we can set the following limits: q min -q p∆T < u < q max -q p∆T (5)

where q min and q max ∈ R n are the angular joint limits, and ∆T is the control loop rate. p > 1 serves as a conservative factor; i.e., the robot will not violate the constraint even if the robot keeps the same velocity over the next p time-steps. Therefore, the higher p, the slower the joints move when they get close to their limits. We choose p = 2. The joint limits are experimentally set to q min = [-∞, -120, -∞, -120, -∞, -120, -∞] and q max = [∞, 120, ∞, 120, ∞, 120, ∞] where ∞ refers to the fully-rotational joints. The inequalities in Eq. 4 and 5 lead to the following QP constraints:

u min = max{-qmax , (q min -q)/p∆T } u max = min{ qmax , (q max -q)/p∆T } (6)
Power safety: Average power consumption of Kinova ® (45W [START_REF] Kinova | User guide: Kinova gen 3[END_REF]) respects ISO norms which defines 80W as the limit for cobots [START_REF]&2:2011, Safety requirements for industrial robots[END_REF]. Furthermore, given the power at the end-effector as τ T u, we consider the following inequality constraint:

-P max < τ T u < P max [START_REF] Xie | Augmenting human with a tail[END_REF] with P max = 4W. This threshold is chosen experimentally by observing the value of τ T u when the robot is operating satisfactorily in terms of resulting velocities and interaction forces. It is important to note that here we use the current torque measurement to estimate the power at the next timestep; i.e., assuming slow dynamics for interaction forces. In other words, the robot regulates its velocities based on the measured interaction forces. Moreover, we only limit the projected power at the end-effector and not for any other specific point on the robot body. Nevertheless, this is not a safety issue since there are other constraints at the joint-level.

Workspace safety: To avoid the collision between the wearable robot and its user, we model the human torso and head using bounding boxes as illustrated in Fig. 2. Here, we only consider collision avoidance with the user's head and torso. In other words, we allow physical interaction of the robot with the user's arms where its safety is handled via other parts of our mid-level controller. Using the QP constraints, we limit the robot's joints from entering these volumes. To this end, each bounding box is defined as a set of points (X min , X max , Y min , Y max , Z min , Z max ) in the base frame. Here we assume that the user's torso and head movement with respect to the base of the robot is negligible considering a conservative choice for the size of the bounding boxes. We constraint the Cartesian velocity of each joint as follows:

x i + ẋi k∆t > x + i if x i > x + i x i + ẋi k∆t < x - i if x i < x - i (8)
where x i and ẋi ∈ R for i = 1...n are the position and velocity of the ith joint of the robot. The upper and lower boundaries x + i and x - i ∈ R 3 are computed by aggregating the two bounding boxes. These constraints can be written as

J i u > (x + i -x i )/(k∆t) if x i > x + i J i u < (x - i -x i )/(k∆t) if x i < x - i (9)
where J i ∈ R n×3 for i = 1...n are the partial Jacobians. These constraints are then added to the QP solver as a part of the A matrix; 3 constraints per joint. Furthermore, to have a conservative and damped behavior near the bounding boxes, we choose k = 20.

The inequalities in Eq. 7 and 9 lead to construction of the A ∈ R 4n×n matrix and the corresponding boundaries for b min and b max ∈ R 4n .

A =      τ T J 1 . . . J n      , b min =      -P min Γ(x + 1 -x 1 )/(k∆t) . . . Γ(x + n -x n )/(k∆t)      (10)
where the switching function Γ(x) = x for x < 0 and a large value otherwise in order to rend the constraint void. The computation for b max is done similarly.

Collision safety: In order to provide a safe and versatile interaction with the environment, we handle the collisions at two levels; namely "soft" and "hard" collisions. Unlike the previous safety factors that were handled by the QP solver, collision safety is handled by the admittance loop. Soft collision is concerned with interaction forces that we expect from a safe physical interaction with the environment and humans; e.g., the human pushes the robot or when the robot physically manipulates an object. For this purpose, we use the following admittance feedback loop.

ẋa = αJ †T Φ(τ -τ g , τ sof t ) ( 11
)
where Φ is a deadzone function that is applied element-wise as follows:

Φ i (τ, τ ′ ) =      τ i -τ ′ i if τ ′ i < τ i 0 if -τ ′ i < τ i < τ ′ i τ i + τ ′ i if τ i < -τ ′ i ( 12
)
where τ ′ ∈ R n is an arbitrary vector with positive values.

In our case, we choose τ sof t = [START_REF] Eden | Principles of human movement augmentation and the challenges in making it a reality[END_REF][START_REF] Hussain | A soft supernumerary robotic finger and mobile arm support for grasping compensation and hemiparetic upper limb rehabilitation[END_REF][START_REF] Hussain | A soft supernumerary robotic finger and mobile arm support for grasping compensation and hemiparetic upper limb rehabilitation[END_REF][START_REF] Hussain | A soft supernumerary robotic finger and mobile arm support for grasping compensation and hemiparetic upper limb rehabilitation[END_REF][START_REF] Parietti | Supernumerary robotic limbs for aircraft fuselage assembly: body stabilization and guidance by bracing[END_REF][START_REF] Parietti | Supernumerary robotic limbs for aircraft fuselage assembly: body stabilization and guidance by bracing[END_REF][START_REF] Hussain | Using the robotic sixth finger and vibrotactile feedback for grasp compensation in chronic stroke patients[END_REF]N m which acts as the threshold below which no collision is detected. Moreover, τ g is the estimated gravity effect and the measured torque on the ith joint respectively. J † is the pseudo-inverse of the Jacobian. Here, α is the admittance gain that controls the behavior of the response (response time, stability, and oscillations); i.e., higher/lower α leads to lower/higher damping behavior. Therefore, in the case of a soft collision, this admittance rule moves the end-effector based on the interaction forces. This admittance loop also allows the user to indirectly control for the interaction forces through ẋt .

F = K v ( ẋt + ẋa ) ẋa = αF (13)
This leads to

F = 1 1/K v -α ẋt (14)
where K v is the forward gain imposed by the velocity controller. There, given high values for K v , in interaction with the environment, -ẋt /α can be applied to the environment. However, this relation is only valid in a steady state while during the transient phases, these forces can be much higher, depending on the inertia and velocity of the robot. Therefore, it is crucial to consider a "hard" collision mode.

Hard collision: this mode classifies the abnormal collisions which might harm the human (or the robot). Such collisions are recognized by a higher level of force/torque (compared to the soft one). The implemented strategy is to stop the task (ignoring the desired end-effector motion) and render the robot fully compliant. To detect hard collisions, at each timestep, we check if any joint satisfies the following criterion: commands to the robot and switch the compliant mode implemented by Kinova ® on the robot. The robot stays in this mode until the user specifies otherwise manually.

|τ -τ g | > τ hard (15)

III. RESULTS AND DISCUSSION

To validate our control architecture, we present two experimental scenarios. In the first scenario, we test the robot motion-planning in its joint space. To do this, we send a constant velocity for the end-effector ( ẋt = [0, .05, .02]) while we expect the robot to avoid the bounding boxes and joint position and velocity limits. These results are illustrated in Fig. 3 with its corresponding joint position and velocity in Fig. 4. Given our choice for the slack variable, the robot is allowed to deviate slightly from the straight line in order to execute the task. Let us note that without the slack variable, the robot stalls when there is no solution for the QP problem. The size the slack variable controls how much the robot's end-effector velocity can deviate from the desired one. This figure shows that not only end-effector but also all joints penetrating into the bounding boxes. behavior allows the user to easily move the robot's effector around (via any interface) without being worried about the robot's configuration and collision. In other the user is not to 1) control all the joints individually, or 2) send precise commands to the end-effector. Furthermore, Fig. 4 illustrates other safety constraints were respected as well; i.e., joint velocities remain in the safe zone, and joint position limits are respected.

In the second experiment, we test the robot's behavior in terms of physical interaction with the environment. To this end, we put the robot's end-effector in physical contact with an external force sensor while we send different desired endeffector velocities to the robot. This result is depicted in Fig. 5 where we slowly increase the desired end-effector Fig. 6. The result of impacts when the robot collide with the environment at different speed. Higher velocities results in higher forces at the impact which is detected as hard collision and trigger the low-level admittance control. The plot of the power shows that this value remains in the boundaries before we switch to low-level admittance. velocity (x t from 0 to 0.2m/s). This increase in the desired velocity leads to an increase in the interaction forces between the robot and the environment. After a certain threshold around F = 90N which depends on τ hard and the robot's configuration) the robot detects a hard collision and switches to the low-level admittance control which ignores the desired task. Thus the interaction forces disappear and the robot becomes compliant which allows the user to move the robot freely.

In the third experiment, we test the effect of impacts; i.e., the robot follows a constant desired velocity and collides with a hard surface. These results are illustrated in Fig. 6 which shows that a similar safety behavior to the previous figure is obtained; i.e., colliding with higher interaction forces triggers the low-level admittance control. The other subplot shows the projected power at the end-effector (τ T q). We can see that before collision, this power remains inside the designated boundaries. The behavior of such power exchanges with the environment after the collision depends on the control mode. For the collisions with higher velocities ( ẋd = 0.15 and 0.2), the low-level admittance control is triggered. We can see that this low-level controller is not the power, however, providing a passive interaction with the environment. However, operating the robot using the QP in a safe range of velocities and interaction forces provide a satisfactory postcollision behavior. Finally, let us note that in the second and third experiments, we used a fixed-base configuration to be able to investigate the controller in physical interactions with rigid contacts. In the worn configuration, the situation is ameliorated given the compliance of the human body and the human ability to displace the robot's base.

IV. CONCLUSION

In this preliminary work, we presented a mid-level control strategy for a safe supernumerary robotic arm. We formulated our control as a constrained inverse kinematic problem that can be solved using Quadratic programming. We focused on two main safety considerations: workspace safety and interaction safety. These considerations were translated to inequality constraints for the QP problem; i.e., constraints on the commanded joint velocities. The upper/lower bounds of these constraints were extracted based on standardized safety norms. We experimentally validated our approach in a controlled laboratory environment. Our results show that such QP problems can be effectively solved which leads to a safe robotic behavior. Furthermore, we presented a generalized formulation that can be easily adapted to different robotic supernumerary arms. We provided guidelines and necessary details for each constraint to improve the reproducibility and adaptability of our controller. In our future work, we will exploit the information about the movement of the humanuser to avoid dangerous or uncomfortable configurations; e.g., the risk of falling when the user bends forward with the robot extended forward as well. Furthermore, the human compensatory movements can be used for intentionrecognition purposes and be used to improve the robot's autonomy and reactiveness; see our previous work on such control strategies [START_REF] Legrand | Closing the loop between body compensations and upper limb prosthetic movements: A feasibility study[END_REF][START_REF] Legrand | Controlling upper-limb prostheses with body compensations[END_REF][START_REF] Khoramshahi | Intent-aware control in kinematically redundant systems: Towards collaborative wearable robots[END_REF][START_REF] Khoramshahi | Identification of inverse kinematic parameters in redundant systems: Towards quantification of inter-joint coordination in the human upper extremity[END_REF].

Fig. 2 .

 2 Fig. 2. View of the head and torso bounding boxes (in transparent green color) which are defined in the frame of the robotic arm base, i.e. the operator trunk.

Fig. 3 .

 3 Fig. 3. The result of robot motion planning in avoiding the designated bounding boxes while following the desired end-effector velocities.

Fig. 4 .

 4 Fig. 4. The corresponding joints position and velocity for Fig. 3. The joint velocity limits are respected and the resulting joint positions are satisfactory in terms of smoothness. No collision or penetration into the bounding boxes occurs in this demonstration.

Fig. 5 .

 5 Fig.5. The resulting behavior with respect to the interaction forces. The robot is initially placed in contact with a hard surface as the desired velocity ( ẋt) increases over time. High interaction forces are recognized as hard collisions where the robot ignores the task and switches to a low-level admittance control.

This work is supported by H2020 FET NIMA project (899626).


domain: sdv info

text:
Maud Alix

Daniel Żarski

Dominique Chardard

Pascal Fontaine

Berenice Schaerlinger

Deformities in newly hatched embryos of Eurasian perch populations originating from two different rearing systems

Keywords: Embryonic deformities, recirculating aquaculture system, Perca fluviatilis, histology, husbandry, rearing systems, embryonic development, Lorraine ponds

During the teleost ontogenesis, biotic and abiotic factors influence organs deformations. Despite of an important literature, the potential causes of deformities in fish are still poorly understood. Cultured fish in artificial conditions exhibit higher incidence of developmental impairments compared to wild ones. This study aims at describing exhaustively the deformity categories of newly hatched embryos of Eurasian perch and investigating at once all biotic and abiotic factors of two extremely different rearing systems [recirculating aquaculture system (RAS) vs. ponds] on their occurrence. The description and clustering of deformities according to the organs involved allowed not only to show that skeletal, cardiac and yolk anomalies are the most common ones, but also to highlight new functions undergoing developmental failure (locomotive, digestive and excretory systems). In total, 10 categories of abnormalities have been described. In addition, our data show a relationship between the cardiac and yolk deformities suggesting common causes. In a second part, we compared the incidence of deformities in the progeny of two distinct populations of fish, reared under extremely various conditions (RAS vs. pond). The total deformity rates did not differ between the groups but the nature of deformities was surprisingly different as the RAS reared fish (RRF) originating from fisheries presented a higher variety of deformity categories than the fish originating from Lorraine ponds (LPRF). Indeed, cardiac, yolk and skeletal axis deformities were more frequent in RRF embryos, whereas mouth abnormalities were preferentially represented in LPRF embryos. These findings may help understanding the potential causes of these developmental impairments in cultured animals and may lead to the improvement of husbandry conditions.

Introduction __________________________________________________________________

During animals life cycle, several morphological, anatomical, physiological or behavioral deviations from a standard phenotype may occur [START_REF] Divanach | Abnormalities in finfish mariculture: an overview of the problem, causes and solutions. Seabass Seabream Cult[END_REF]. Therefore, it derives from various intrinsic and/or extrinsic factors contributing to the decrease in the developmental success, especially for the early life stages during which the definitive phenotype is built [START_REF] Balon | Epignesis of an epigeneticist :the development of some alternative concepts on the early ontogeny and evolution of fishes[END_REF]. In that context, the occurrence of divergent phenotypes during the early development can increase the susceptibility of animals to predation and could lead to the decline and the extinction of a population [START_REF] Westernhagen | Developmental defects in pelagic fish embryos from the western Baltic[END_REF]. The complex issue of understanding the potential causes of developmental impairments needs detailed investigations to reveal new processes potentially disturbed during the breeders life history and the offspring development [START_REF] Divanach | Abnormalities in finfish mariculture: an overview of the problem, causes and solutions. Seabass Seabream Cult[END_REF][START_REF] Bonnet | Microarray-based analysis of fish egg quality after natural or controlled ovulation[END_REF][START_REF] Pulcini | Testing the relationship between domestication and developmental instability in rainbow trout, Oncorhynchus mykiss (Teleostei, Salmonidae)[END_REF]. This is important in farming animals where the production is particularly affected by these problems and in wild populations submitted to environmental changes [START_REF] Divanach | Abnormalities in finfish mariculture: an overview of the problem, causes and solutions. Seabass Seabream Cult[END_REF][START_REF] Réalis-Doyelle | Strong effects of temperature on the early life stages of a cold stenothermal fish species, Brown Trout (Salmo trutta L.)[END_REF].

Most of studies aimed at determining the effect of some specific abiotic or biotic factors on the developmental impairments, especially in species with external development as most of the finfish that are sensitive to the external factors. In that context, the effects of specific environmental factors [START_REF] Westernhagen | Developmental defects in pelagic fish embryos from the western Baltic[END_REF][START_REF] Jezierska | The effects of heavy metals on embryonic development of fish (a review)[END_REF][START_REF] Villamizar | Effects of light during early larval development of some aquacultured teleosts: a review[END_REF][START_REF] Bondarenko | Effect of water temperature on egg incubation time and quality of newly hatched larvae of northern pike (Esox lucius L., 1758)[END_REF][START_REF] Réalis-Doyelle | Strong effects of temperature on the early life stages of a cold stenothermal fish species, Brown Trout (Salmo trutta L.)[END_REF], nutritional factors [START_REF] Fernández | Larval performance and skeletal deformities in farmed gilthead sea bream (Sparus aurata) fed with graded levels of Vitamin A enriched rotifers (Brachionus plicatilis)[END_REF][START_REF] Boglione | Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors[END_REF] and husbandry practices (Aegerter & Jalabert, 2004) on the occurrence of deformities have already been reported for several species. However, these studies focused on the global deformity rates and several categories of deformities as the blastomeres cleavage, the skeletal axis, jaw, swim bladder or yolk deformities [START_REF] Johnson | Mortality, growth and swim bladder stress syndrome of sea bass (Dicentrarchus labrax) larvae under varied environmental conditions[END_REF][START_REF] Andrades | Skeletal deformities in larval, juvenile and adult stages of cultured gilthead sea bream (Sparus aurata L.)[END_REF] As a whole, a high prevalence of deformed fish in captive conditions have been reported [START_REF] Koumoundouros | Morpho-anatomical abnormalities in Mediterranean marine aquaculture[END_REF][START_REF] Boglione | Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors[END_REF]. Considering the extreme, freshwater fish can be reared in ponds, close to the wild conditions, or in recirculating aquaculture system (RAS) in which all conditions are controlled to optimize fish growing and reproduction. As a consequence, cultured fish can confront various types of environments depending of the rearing system including the lack of predators, food, high density of fish and sometimes inappropriate captive conditions influencing several biological functions among which the reproduction [START_REF] Andrades | Skeletal deformities in larval, juvenile and adult stages of cultured gilthead sea bream (Sparus aurata L.)[END_REF][START_REF] Pulcini | Testing the relationship between domestication and developmental instability in rainbow trout, Oncorhynchus mykiss (Teleostei, Salmonidae)[END_REF]. In addition, fish maintained in one specific rearing condition are usually adapted to it. In other words, comparing fish reared in pond or RAS conditions involves to take into account not only all environmental modifications but also intrinsic factors of the breeders. Up to now, no study aimed at investigating the global effect of all these extrinsic and intrinsic parameters at once on the deformities occurrence in the progeny.

The Eurasian perch, Perca fluviatilis L., is a promising and valuable species for the diversification of freshwater aquaculture in Europe. It is reared in RAS and pond systems and is considered at a level 4 of the domestication process according to Teletchea & Fontaine (2014). Despite of the control of its reproduction cycle, the incidence of developmental impairments is still variable [START_REF] Schaerlinger | Evaluation and improvements of egg and larval quality in percid fishes[END_REF]. The Eurasian perch is an interesting species to study due to its high fecundity, a detailed description of its normal embryonic development [START_REF] Alix | An alternative developmental table to describe nonmodel fish species embryogenesis: application to the description of the Eurasian perch (Perca fluviatilis L. 1758) development[END_REF], and the easiness to observe early developmental impairments thanks to the egg envelope transparency. Moreover, the perch spawn can easily be found from various origins (Zarski et al., 2016).

The present study aims at (1) investigating the morphological deformities in newly hatched embryos and (2) identifying the potential relationships between these abnormalities to (3) determine the global effect of all extrinsic and intrinsic factors of populations reared in two extreme rearing conditions (pond vs. RAS) on the occurrence of deformities in Eurasian perch.

Material and Methods __________________________________________________________

Broodstock and eggs management

Fish were handled in accordance with national and international guidelines for the protection of the animal welfare (Directive 2010/63/EU). The study was conducted during consecutive years (2013, 2014 and 2015). Fish originated either from Lorraine ponds (LPRF, three batches of spawn) or a fishfarmer broodstock reared in RAS conditions (RRF, two batches) (Table 1).

Rearing protocol of LPRF

Lorraine pond reared fish were caught 1 month before the spawning season and transferred to outdoor facilities (tanks of 3000 L, Lorraine, France) to facilitate their manipulation. At that time, females were about to ovulate and males were slightly spermiating. During the spawning season, males and females were kept together in tanks, the photothermal conditions were natural to promote spontaneous oocytes maturation and water temperature records varied between 8 and 16°C. Once the first female spawned, each of them was checked daily to determine the ovulation time. The day of their ovulation, females were hand-striped into a dry container prior to the in vitro fertilization.

Rearing protocol of RRF

Recirculating aquaculture system reared fish provided by a fish farm (Lucas Perches, Lorraine, France), were reared in an intensive system and fed with commercial pellets (Sturgeon Grower, Le Gouessant) since the early juvenile stage and transferred to our experimental facilities until the beginning of the experiments. Males and females were kept together in 500 L (2013) or 2000 L (2015) tanks connected to a RAS, with an automatic system controlling the temperature and photoperiod [START_REF] Fontaine | Broodstock management and control of the reproductive cycle[END_REF]. Following the induction of the gonadal cycle, fish were led to mature and spawn without any additional treatment but environmental stimulation (increment of the temperature up to 12°C and dark/light photoperiod up to 8:16). Once a female was recognized to be ready to spawn, the eggs were stripped manually as described above.

In vitro fertilization and incubation of the embryos

For each fish group, the same spawning protocol was applied.

The fertilization was performed according to the 'wet method' (Zarski et al., 2012). A mix of sperm from three males was added to each spawn (0.10 ml sperm.g-1 dry eggs). During the embryogenesis, each spawn was incubated uninterruptedly until hatching at 13.0 f 0.5°C in a hatchery with automatic thermoregulation and UV sterilization. A constant dark/light photoperiod (8:16) was applied at a light intensity of 200 lx at the water surface. For each spawn, three samples (about 100 eggs per sample) were incubated separately to perform the experiment the same way on each sampling. The water quality of breeder's tanks and incubator are summarized in Table S1.

Characterization and occurrence of deformities

The embryonic period was considered from the fertilization to the onset of exogenous feeding according to [START_REF] Alix | An alternative developmental table to describe nonmodel fish species embryogenesis: application to the description of the Eurasian perch (Perca fluviatilis L. 1758) development[END_REF].

Observations of living embryos

Each spawn was monitored daily during the hatching period to collect embryos as soon as they hatch. Embryos captured on the day of their hatching were observed immediately using a stereoscopic microscope (Olympus, SZX7) at low magnification (92) to evaluate the presence of deformities. They were called newly hatched embryos (NHE). We checked several organs, such as the skeleton axis, mouth, eyes, yolk, fins-related structure (pectoral fins anlagen and finfold), digestive and cardiac system, that are easily visible with this technique. To investigate more accurately which organs present an abnormality and describe the impairments, each abnormal individual was photographed the same day using a light upright optical microscope (Nikon Eclipse Ni-U) associated with a DS-Fi2 digital camera and the software NIS BR (Nikon France, France) at low magnification (920).

Abnormalities occurrence was certified after a careful comparison with normal NHE morphology at the same age [START_REF] Alix | An alternative developmental table to describe nonmodel fish species embryogenesis: application to the description of the Eurasian perch (Perca fluviatilis L. 1758) development[END_REF]. Each NHE was assigned to one of 10 categories according to the organs involved [axis (Ad), cardiac (Cd), yolk sac (Yd), mouth (Md), eyes (Ed), digestive system (Dd), fins-related structure (Fd), urinary system (Ud), head (Hd) or multivariable deformed (MVd)]. Some embryos exhibited several deformations in various organs. They were taken into account in each category (except embryos exhibiting MVd).

Histological study

Deformed embryos were fixed the same day in Bouin's solution for 48 hours, prepared, cut and stained as described in [START_REF] Alix | An alternative developmental table to describe nonmodel fish species embryogenesis: application to the description of the Eurasian perch (Perca fluviatilis L. 1758) development[END_REF]. Stained sections were examined using a light upright optical microscope (Nikon Eclipse Ni-U) at 9100 magnifications (Nikon France, Champigny-sur-Marne, France). To better characterize deformities involving the cardiac system, yolk, mouth and skeletal axis (abnormalities more often observed), five embryos per organ coming from 15 different spawn were longitudinally cut. In addition, five embryos were transversally cut for the cardiac and yolk deformities.

Data analyses

In total, 42 spawn were examined (30 from RRF and 12 from LPRF) among which 585 NHE exhibit macroscopic deformities (Table 1). For each spawn, the total number of NHE were taken into account to calculate the total deformity rate (Dr=number of deformed embryos/number of hatched embryos 9 100, in %). The mean deformity rate has been obtained for each of the fish group f SE (RRF and LPRF). In addition, specific deformity rates were calculated as the number of hatched embryos exhibiting a particular deformity in relation to the total number of abnormal embryos (in %). The assumption of normality (Shapiro test) and homogeneity of variances (Levene test) were tested for all the deformities variables. The specific deformities rates were represented with box plots in RRF or LPRF. When data did not respect the assumption of normality, anon-parametric Mann-Whitney test was used to determine a significant difference between the total deformities or specific deformities rates according to the global effect of abiotic and biotic factors linked to the rearing conditions. Statistical analyses were performed on the mean of deformity or specific deformities rates.

In order to determine potential relationships between categories of deformities, a principal component analysis (PCA) was performed using the specific deformities rates and represented on a correlation circle. Moreover, a Pearson's correlation table was generated. Data analyses were performed with "R" software (version 3.2.5; The R Foundation for Statistical Computing, Vienna, Austria).

Results ______________________________________________________________________

Investigating the deformities in newly hatched embryos (NHE) allows not only characterizing deformities commonly described in other species and in various environmental conditions, but also less described categories.

Macroscopic and histological descriptions of embryonic deformities

Axis deformities

Axis deformities were found to be the most frequent category of impairment (61.1 f 4.9%). Among them, one distinguishes lordosis (V-shaped dorsal-ventral curvature), kyphosis (Ashaped dorsal-ventral curvature) and scoliosis (lateral curvature) (Fig. 1b). The histological analysis did not reveal any cellular defect in the notochord or the tissues surrounding the deformed parts of the body (e.g. muscle).

Mouth deformities

Mouth deformities were the second most frequent deformity category recorded (33.6 f 4.8%). They were primarily observed on the lower jaw, with a shortening (Fig. 1c,d), elongation ora gaping mouth phenotype (Fig. 1e). No difference between normal and abnormal tissues was found at the histological level.

Yolk deformities

Yolk deformities represent 27.8 f 4.5% of the deformed embryos. NHE exhibit mainly yolk sac edema, pear-shape yolk or yolk detachment (Fig. 1b,c,e, star). This yolk detachment is accompanied by an abnormal morphology of the liver which is drawn out between the intestine and the yolk. In comparison with normal specimens (Fig. 2a,c,e), the histological study reveals that NHE with yolk edemas exhibit generalized edema in the body as described more accurately below (Fig. 2b,f).

Cardiac deformities

Two main types of deformities have been observed. First, cardiac cavity edemas (Fig. 1b,c,e), secondly, a modification of the heart shape which is elongated and to a less extent heart atrophy (Fig. 1c,e). Cd were found in 27.3 f 4.7% of deformed embryos. As previously mentioned, cardiac and yolk edemas often occurred simultaneously (85% of the embryos exhibiting an edema). The histological study revealed also edemas in the head, upper jaw, around the eyes, under the esophagus and between organs of these NHE (Fig. 2b,d,f). In addition, it corresponds to the presence of loose connective tissue (Fig. 2d) and empty spaces observable around the pectoral fins anlagen (Fig. 2f).

Eyes deformities

Eyes deformities were found in 8.7 f 2.9% of the deformed embryos and could be subdivided into two main types. First, a total or partial fusion of the optic cup (cyclops) and second a pigmentation-deficient eyes (Fig. 1d,e). Moreover, the histological analyses showed that Ed could be observed on transversal or sagittal sections of embryos that did not present any macroscopic eyes deformities (Fig. 3). Nevertheless, it may coincide with other categories of deformities as generalized edemas. In these conditions, slight or total disorganization of the cellular layers (Fig. 3b,c,d,f) could be seen compared to the normal eyes structure (Fig. 3a). Actually, bipolar, ganglion and amacrine cells blended in the same cellular layer instead of three distinct ones (Fig. 3b,c). Moreover, the choroidal melanocytes layer was damaged at various levels and detached from the other ones (Fig. 3c). In more severe cases, a total disorganization of all cell layers especially for the choroidal melanocytes was observed (Fig. 3d,f). In some cases, a fusion of the optics cups with the merge of several layers was observed (Fig. 3e), even in embryos without any macroscopically visible eyes impairments. Finally, the NHE presenting a partial fusion of the eyes macroscopically, showed a merged optic cups with a complete disorganization of cellular structure (Fig. 3f).

Digestive system deformities

Digestive system deformities were found in 3.5 f 1.3% of the deformed embryos. Various types of abnormalities have been observed among which the digestive tract hypertrophy (Fig. 4b,c,d,g), clearly visible in the intestine with different scales of severity (Fig. 4b,c,d) and could be observed in the rectum (Fig. 4c,d,g). Digestive hypertrophy was often associated with the presence of other edemas (Fig. 4b,c,g,h). The second kind of deformities corresponds to an absence of folded intestinal villi (Fig. 4e) potentially leading to a decrease in the absorption surface. In spite of this, the mucosal folding pattern was visible in the rectum.

Fins-related structure deformities

Fins-related structure deformities correspond to 2.0 f 0.7% of the deformed embryos. They were either associated to the anal orifice absence and then mainly correspond to a continuous veil in the ventral part of the NHE (Fig. 4b,c,f,g). Another type of fins deformities is a lack of development of the pectoral fins anlagen (data not shown).

Urinary system deformities

Urinary system deformities correspond to 1.6 f 0.9% of the deformed embryos. These abnormalities correspond mainly to the absence of the urinary bladder (Fig. 4b,c,d,f,g). Rarely, hypertrophies of the posterior part of pronephric ducts and urinary bladder were noticed (Fig. 4h). As for the digestive system, the hypertrophy was often associated with the presence of edemas (Fig. 4h). Finally, an absence of urinary or anal orifice was observed after the rectum and the urinary bladder (Fig. 4f).

Head deformities

This designation mainly groups abnormalities involving defects of central nervous system or the sensitive organs placed on the head. For example, a displacement of the olfactory organs was observed (data not shown). They correspond to 1.1 f 0.7% of the deformed embryos.

Multivariable deformities

Finally, this category involves NHE lacking a large part of the normal body. For example, when the anterior or the posterior parts of the embryos are lacking, it will lead to individuals without the head or the tail (data not shown). These abnormalities are rare with 1.1 f 0.6% of the deformed embryos.

Potential relationships between the categories of deformities

In every spawn, most of the NHE exhibit multiple deformities.

The next question was thus to determine whether some of them always occur in the meantime, suggesting some common causes and/or mechanisms impaired. To do so, correlations were performed between the specific deformities rates of each category (Fig. 5 and Table 2). Firstly, three defined axes explained 61.9% of the variance (29.8, 16.5 and 15.6% for the axis 1, 2 and 3, respectively) (Fig. 5). The first axis was strongly associated to the Yd and Cd (r = 0.91 and r = 0.87, respectively). The second axis was mainly described by the Md (r = 0.62), whereas the third axis was correlated with the MVd and Ud (r = 0.83 and r = 0.65). As these data suggest that predominant relationships could exist between deformities categories linked to the same axis, Pearson's correlation coefficient values were generated, indicating that only the Yd and Cd were highly and significantly correlated (r = 0.84, Table 2).

Incidence of deformities categories according to the global effect of biotic and abiotic factors linked to the rearing system

This work shows that the Øand LPRF present various developmental impairments. Within a spawn, several categories of abnormalities were observed and numerous embryos presented several deformities. The total deformity rate (Dr) ranged from 0.8 to 20.8%, depending on the spawn. No significant difference was observed in the Dr according to the broodstock groups and mean values amounted to 6.2 f 0.99% and 5.8 f 1.66% for the RRF and LPRF, respectively. In addition, the deformity categories with an occurrence lower than 3.5% (Dd, Fd, Ud, Hd and MVd), varied between 0 and 30% according to the spawn. The deformities with occurrences higher than 8.5% (Ad, Cd, Yd, Md) varied between 0 and 100%. A detailed statistical analysis revealed that the Md and Ad correspond to the main deformities found in LPRF fish, while the RRF present a higher variety of organs deformed (Fig. 6a,b). Moreover, Ad, Cd and Yd were significantly higher in RRF than in LPRF (P < 0.05) (Fig. 6c,d,e). Interestingly, the Md was more often frequent in LPRF than RRF (Fig. 6f). No significant difference was found between the LPRF and RRF for other categories of deformities.

Discussion ___________________________________________________________________

Enhancing the knowledge of embryonic deformities helps to improve the rearing conditions. Current works mainly focused on the effect of some specific intrinsic/extrinsic factors on some specific deformities. These studies did not take into account the potential interactions between all factors that could lead to deformities occurrence. As a starting point, we thus compared two populations of fish adapted to extremely variable environments. We thus took into account all abiotic and biotic factors varying in the two conditions as a whole. As a consequence, several hypotheses could explain differences between the two populations as the feeding regime, the genetic, especially family structure or consanguinity levels [START_REF] Afonso | Association of a lordosis-scoliosis-kyphosis deformity in gilthead seabream (Sparus aurata) with family structure[END_REF][START_REF] Izquierdo | Effect of broodstock nutrition on reproductive performance of fish[END_REF][START_REF] Boglione | Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors[END_REF] as well as other factors. Our exhaustive approach allowed highlighting various developmental impairments depending on the rearing conditions.

So far, it was generally accepted that artificial rearing conditions may have a significant impact on the occurrence of developmental impairments in several species and that the deformities incidence appears to be significantly lower in wild populations [START_REF] Koumoundouros | Morpho-anatomical abnormalities in Mediterranean marine aquaculture[END_REF][START_REF] Boglione | Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors[END_REF]. Nevertheless, here, we did not find any significant differences in the Dr between the Øand LPRF even though a large number of parameters were different between these populations. This may stem from the fact that deformities can appear throughout the fish's life and the difference of deformity occurrence related to rearing conditions could emphasize later. Even if the mean Dr appear low, huge variations occur between the spawn even within a group of fish depending on the organs or systems involved. This extreme variation has huge consequences on the offspring survival or health.

Among the several types of deformity studied in the literature, those involving the skeleton (body axis and mouth), the yolk and the swim bladder are more often described and now well defined for several species [START_REF] Boglione | Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors[END_REF]. Here, we show that in perch also, the main deformities categories observed correspond to the skeletal axis and the mouth deformities as well as the cardiac and yolk deformities. Nevertheless, these last categories were more significantly frequent in the RRF than in LPRF.

Concerning the yolk and cardiac anomalies, we found that they were usually associated together and, to a larger extent, to global edemas. It was already observed in the Atlantic halibut (Hippoglossus hippoglossus) larvae where it was probably related to unfavorable temperatures without any clear link to the broodstock origin [START_REF] Ottesen | Combined effects of temperature and salinity on development and survival of Atlantic halibut larvae[END_REF]. Relationships between broodstock rearing conditions and yolk abnormalities was reported in Atlantic salmon (Salmo salar), because of an excessive stress during the pre-spawning period [START_REF] Eriksen | Prespawning stress in farmed Atlantic salmon Salmo salar: maternal cortisol exposure and hyperthermia during embryonic development affect offspring survival, growth and incidence of malformations[END_REF]. It can thus be suggested that higher incidence of yolk deformities observed in this study in the case of RRF could be related to the stress level experienced by the breeders.

Conversely to the deformity categories previously mentioned (e.g. Ad, Cd, Yd), the incidence of Md was significantly higher in NHE originating from LPRF than from RRF. Little information is available about the potential determinants of this type of deformity, except the nutrition and the genetics [START_REF] Boglione | Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors[END_REF]. Here, the significantly high mouth deformity frequency in LPRF compared to RRF NHE may be explained by pollutants potentially present in the pond system [START_REF] Gaillard | Rôle des étangs de barrage à vocation piscicole dans la dynamique des micropolluants en têtes de bassins versants[END_REF]. Indeed, the accumulation of environmental contaminant in fish oocytes may affect the developmental success and provoke mouth deformities [START_REF] Jezierska | The effects of heavy metals on embryonic development of fish (a review)[END_REF].

Ed were observed in some species but have poorly been studied. It has been shown that they can result from several factors (e.g. ultraviolet light exposure, post-ovulatory aging, nutrition and rearing conditions) and can impact fish health, behavior, survival and growth [START_REF] Bonnet | Microarray-based analysis of fish egg quality after natural or controlled ovulation[END_REF]McElwain et al., 2013). Our histological studies reveal various level of cellular layers disorganization.

Interestingly, some macroscopically normal eyes exhibit abnormalities in the cellular structure. They may lead to individuals with huge visual impairments, potentially compromising their survival as the Eurasian perch is a visual predator [START_REF] Vlavonou | Elevage experimental de la perche Perca fluviatilis L.: développement larvaire et croissance[END_REF]. In order to further investigate these deformities, it would be interesting to couple more detailed cellular structure of the eyes study with behavioral experimentations. It would allow understanding the consequences of these structural anomalies on the visual and biological responses of the larvae and potentially explain some lethalities observed in hatcheries by the time of first exogenous feeding.

Other categories of deformities (digestive, fins, urinary, head and multivariable deformities) have never or very poorly been described in the literature. It may be due to their little frequency and difficulties in observation compared to other anomalies. However, they may explain, at least partly, some survival troubles at some specific stages. These anomalies may alter some important biological functions such as the digestion, excretion and fish locomotion. The results observed in this study indicate that the fin-related-structure deformities, widely observed in the perch hatcheries, may stem from the impairments determined already at the embryonic stages. According to the normal perch developmental table [START_REF] Alix | An alternative developmental table to describe nonmodel fish species embryogenesis: application to the description of the Eurasian perch (Perca fluviatilis L. 1758) development[END_REF], the anus closure or urinary bladder development always occur at the same time (7 dpf for the urinary bladder and 10 dpf for the anus opening). In the present study, embryos were collected at their hatching date (6-15 dpf) and compared to normal embryos of the same age. As a consequence, in our case, these variations rather reflect developmental defects than normal variations in the chronology as it could happen in other fish species. Further works are needed to better characterize all these developmental abnormalities.

As a conclusion, this study is the first one characterizing various deformities categories according to the rearing conditions of breeders. For the freshwater fishes, the pond and RAS correspond to the extreme opposite rearing conditions in term of human control of the fish environment. In addition, fish populations adapted to these conditions present phenotypic and genetic divergences that could also influence the deformities occurrence. In the literature, most of studies tented to understand the effect of individual factors on the deformities occurrence. However, the combination of several parameters may have additive or antagonist effects. Taking into account in the meantime all factors allows making a state of place of the differences occurring between the two rearing systems. Even if some of the abnormalities may be related to some specific factors, further studies are needed to improve our knowledge and the fish rearing conditions in fishfarms . (e) Cardiac deformities rates (in %). (f) Mouths deformities rates (in %). Some NHE present multiple deformities and are thus included in the counting for several organs. The bold black line represents the median deformities rates, the small black rectangles are the mean specific deformities rates and the small gray rectangles represent atypical spawn with specific deformities rates (outliers or observations considered as abnormal in the population). Different letters indicate significant differences between rearing conditions of reproductive cycle at P < 0.05 using anonparametric Mann-Whitney test. LPRF, Lorraine pond reared fish; RAS, recirculating aquaculture system; RRF, RAS reared fish

Figure 1 .

 1 Figure 1. Description of the most frequent deformities in NHE. (a) Normal NHE. (b) NHE with an axis deformity, kyphosis in the anterior part and lordosis in the posterior part of the body, (black arrowhead) and an edema in the cardiac cavity and around the posterior intestine (stars) leading to the deformation of the yolk (pear-shape yolk). (c) NHE with a mouth deformity with a shortening of the lower jaw (dotted arrow) and a yolk and cardiac edema (stars). (d) Detail of a NHE's head with eyes (large arrow) and jaw deformities (dotted arrow). (e) Anterior part of an abnormal NHE with eyes deformity (large arrow), gaping jaw deformity (dotted arrow, the anterior cartilage bent down and the mouth could not be closed) and multiple edemas (stars). Cc, cardiac cavity; E, eyes; En, encephalon; Ff, finfold; Int, intestine; J, jaw; Ld, lipid droplet; Myo, myotome; Nt, notochord; Y, yolk. Scale bars represent 1000 lm for a-c and 500 lm for d, e.

Figure 2 .

 2 Figure 2. Histological description of NHE presenting cardiac and yolk edema. (a) Sagittal section of the head and the trunk of a normal NHE. Vertical black lines represent the location of transverse sections of other embryos shown in c and e. (b) Sagittal section of the head and the trunk of a NHE presenting a generalized edema. The epithelium is detached from the connective tissue or the underlying myotome in the head and the upper jaw. This edema is also visible in the ventral part of the esophagus, in the cardiac cavity, around the yolk sac and in the ventral part of the tail (black arrowheads). Vertical black lines represent the location of transverse sections of similar other embryos shown in d and f. (c, d) Transverse section presenting the region of the cardiac cavity of (c) a normal NHE and (d) a NHE presenting a generalized edema (black arrowheads) with an epithelial detachment as well as loose connective tissues between the notochord, the esophagus and the mass of the myotomes. The cardiac cavity is elongated because of this edema. (e, f) Transverse section at the level of the trunk of (e) a normal NHE and (f) a NHE presenting a generalized edema (black arrowheads) with an epithelial detachment around all the tissues. Cc, cardiac cavity; E, eyes; En, encephalon; Es, esophagus; H, heart; Int, intestine; J, jaw; Lct, loose connective tissue; Ld, lipid droplet; Li, liver; Mob, medulla oblongata; Myo, myotome; Nt, notochord; Pa, pancreas; Pf, pectoral fin; Ph, pharynx; Sb, swim bladder; Y, yolk. Scale bars represent 500 lm for a-b and 100 lm for c-f.

Figure 3 .

 3 Figure 3. Histological description of several kinds of eyes deformities in NHE. (a) Sagittal section of a normal eye in NHE. (b, c) Sagittal section of abnormal eyes with a blending between several cellular layers as interneuron bipolar (Bc), ganglion (Gc) and amacrine cells (Ac). (b) Slight disorganization with a blending between the amacrine, ganglion and bipolar cells layers. Bipolar cells are visible in the amacrine and ganglion cells layers. (c) Severe disorganization in the amacrine, ganglion, bipolar cells and choroidal melanocytes layers. Bipolar cells are visible in the amacrine and ganglion cells layers. Moreover, the choroidal melanocytes layer is detached from the other ones. (d) Sagittal section of an abnormal eye with a total disorganization of all cellular layers. (e) Transversal section of NHE's head with merged eyes combined with edema inducing epithelial detachment all around the head. (f) Sagittal section of merged eye with total disorganization of cellular layers except Op, On and Vc layers and potential encephalon deformity. Some cellular layers are not visible in this case. Ac, amacrine cells; Bc, bipolar cells; C, cartilage; Cm, choroidal melanocytes; Gc, ganglion cells; Ip, inner plexiform; L, lens; On, outer nuclear layer; Op, outer plexiform; Vc, visual cells. Scale bars represent 100 lm.

Figure 4 .

 4 Figure 4. Description of the digestive, urinary and fins-related-structures deformities in NHE. (a) Normal digestive and urinary system in a NHE. (b-d) Different levels of hypertrophy of the digestive system. (b) NHE with a slight hypertrophy of the intestine (white arrowhead) combined to cardiac edema. (c) Hypertrophy of the intestine (white arrowhead) and the rectum combined to cardiac edema. (d) Severe hypertrophy of the intestine (white arrowhead) and the rectum (black arrowhead) with an axis deformity. (e) Absence of primary folds in the intestinal mucosa (dotted arrow). (f) Absence of the urinary and anal orifice (large arrow). (g) Hypertrophy of the rectum (black arrowhead) and absence of a visible urinary bladder combined with a yolk edema. (h) Hypertrophy of urinary ducts and bladder (black arrowhead) combined with a yolk edema. Cc, cardiac cavity; E, eyes; En, encephalon; Ff, finfold; Int, intestine; Ld, lipid droplet; Myo, myotome; Nt, notochord; R, rectum; Ub, urinary bladder; Y, yolk. Scale bars represent 500 lm.

Figure 5 .Figure 6 .

 56 Figure 5. Study of the potential correlations between the categories of deformities. Correlation circle of the principal component analysis (PCA) presenting the potential relationships between the types of deformities observed in all NHE.

Table 1 . Characteristics of fish groups with the names, their rearing conditions and number of samples used in this study

 1

	up	Origin	Environmental control of	Fish farm	Period of	Number of	Total	Deformed
			the reproductive cycle		spawning	spawn	embryos	embryos
						studied
	RRF	RAS fish	Artificial	Lucas Perches	April 2013	7	9133	382
		farm			June 2015	23
	LPRF Lorraine	Natural	GAEC Piscicole	April 2013	1	3515	203
		ponds		du Saulnois
				GAEC Piscicole	March 2014	5
				du Saulnois
				GFA du	March 2014	6
				Kuhweg
	LPRF, Lorraine pond reared fish; RRF, recirculating aquaculture system reared fish.

Table 2 . Pearson's correlations table on the deformities rates in all newly hatched embryos

 2

		Ad Ed	Yd	Cd	Md	Ud	Dd	Fd	Hd	MVd
	Ad	0.23 0.40 0.3 -0.58 -0.08 0.09 0.17 0.22 0.09
	Ed		0.53 0.45 -0.25 -0.06 -0.05 -0.04 -0.03 -0.10
	Yd			0.84 -0.17 0.13 0.34 0.37 0.30 -0.10
	Cd				-0.03 0.25 0.36 0.43 0.26 -0.04
	Md					0.07 0.04 -0.04 0.06 -0.07
	Ud						0.04 0.14 -0.06 0.54
	Dd							0.47 0.03 -0.06
	Fd								0.26 -0.10
	Hd									-0.07
	Ad, axis deformities, Cd, cardiac deformities, Dd, digestive deformities, Ed, eyes deformities, Fd, fins deformities, Hd, head deformities, Md, mouth deformities, MVd, multivariable
	deformities, Ud, urinary deformities, Yd, yolk deformities. The bold number corresponds to the highly correlated variables (r > 0.80).

Acknowledgements ____________________________________________________________

We thank the French Ministry of Research for M. Alix fellowship and INRA for their financial support of the project. We also thank Yannick Ledor e and Alain Iur etig for taking care of the fish. Finally, we thank LUCAS PERCHES, the GAEC Piscicole du Saulnois and the GFA du Kuhweg for the fish.