Justin Bescop

Nicolas Goeman

Amel Aissaoui

Benjamin Allaert

Jean-Philippe Vandeborre

SMART-RD: Towards a Risk Assessment Framework for Autonomous Railway Driving

Keywords: Autonomous Driving, Dynamic Risk Assessment, Deep learning, Railway Dataset

While the automotive industry has made significant contributions to vision-based dynamic risk assessment, progress has been limited in the railway domain. This is mainly due to the lack of data and to the unavailability of security-based annotation for the existing datasets. This paper proposes the first annotation framework for the railway domain that takes into account the different components that significantly contribute to the visionbased risk estimation in driving scenarios, thus enabling an accurate railway risk assessment. A first baseline based on neural network is performed to prove the consistency of the risk-based annotation. The performances show promising results for vision-based risk assessment according to different levels of risk.

INTRODUCTION

Autonomous vehicles aim to provide many benefits, such as reducing road accidents, congestion, air pollution and improving transport efficiency. These vehicles can move without human intervention, using a combination of sensors, algorithms and communication systems to detect obstacles, assess driving situations and make decisions accordingly. Autonomous car technology has grown significantly, making remarkable advances in road safety, connectivity and mobility [START_REF] Van Brummelen | Autonomous vehicle perception: The technology of today and tomorrow[END_REF]. However, few advances have been made in autonomous trains.

Autonomous vehicle safety is a set of safety protocols, procedures and guidelines which aim to ensure that autonomous vehicles are safe for users, passengers and pedestrians. They relies on sensors and technologies such as cameras, radars and lidars in order to collect data from the surrounding [START_REF] Vargas | An Overview of Autonomous Vehicles Sensors and Their Vulnerability to Weather Conditions[END_REF][START_REF] Zhang | Perception and sensing for autonomous vehicles under adverse weather conditions: A survey[END_REF]. The collected data is then used by sophisticated learning algorithms so as to guide the vehicle autonomously on roads by assessing the surrounding based on many criteria such as obstacle detection, lane conditions and road user behavior anticipation [START_REF] Guo | Is it Safe to Drive? An Overview of Factors, Metrics, and Datasets for Driveability Assessment in Autonomous Driving[END_REF]. It is necessary to develop, behind these sensors, an information pro-cessing chain, usually based on Artificial Intelligence [START_REF] Kuutti | A Survey of Deep Learning Applications to Autonomous Vehicle Control[END_REF][START_REF] Grigorescu | A survey of deep learning techniques for autonomous driving[END_REF], which allows the autonomous system to ensure relevant decision making as a replacement for the human driver, even in an "uncertain" environment.

Datasets are essential for the development of learning-based decision-making systems. For the autonomous car, several datasets are available, which helps the emergence of these systems [START_REF] Janai | Computer vision for autonomous vehicles: Problems, datasets and state of the art[END_REF]. However, the ownership of railways by industrial actors tends to increase the difficulty of designing datasets to evaluate driving systems for the autonomous train. Existing work mainly focuses on improving the performance of low-level estimates such as lane detection or semantic segmentation [START_REF] Yurtsever | A Survey of Autonomous Driving: Common Practices and Emerging Technologies[END_REF]. The hypothesis under such efforts is that these low level estimations are the basis of human drivers' high level driving operations. However, predicting driving commands or suggestions from these low-level estimates has not been well studied. At present, only a few datasets are available for the railway domain, and of these, none is annotated in terms of risk assessment [START_REF] Pappaterra | A Systematic Review of Artificial Intelligence Public Datasets for Railway Applications[END_REF].

In this paper, we propose a new annotation framework for the railway domain containing vision-based annotated risk assessment data, called SMART-RD (riSk assessMent frAmework foR auTonomous Railway Driving). The annotations were performed on the existing RailSem19 dataset [START_REF] Zendel | RailSem19: A Dataset for Semantic Rail Scene Understanding[END_REF], which is widely used for train scene understanding algorithms in the railway domain. This dataset offers a wide range of situations which are very suitable for risk assessment. As illustrated in Fig. 1, the situations are annotated according to three levels of risk, e.g., secure, moderate or high. These levels are defined by a panel of three annotators, according to several criteria: the context (rail profile, track protection), the users (behavior, distance, density), the weather, and the luminosity. Several evaluations have been carried out to highlight the quality of the collected annotations, and the feasibility of training a decision making system based on these data.

The paper is structured as follows: Section 2 presents a position on the development of datasets to train decision making systems to assess a risk for autonomous driving. The design of the framework, from the annotation protocol to the study of the collected annotations, is given in Section 3. Section 4 contains the evaluation of a first decision making system based on our framework. A conclusion is made in section 5.

RELATED WORKS

Decision-making algorithms need to be trained and evaluated on datasets in order to get a model with a high generalization and to ensure the safety of the system. To our knowledge, there are no annotated datasets on risk assessment based on a vision system in the railway domain. Therefore, the present analysis is based on datasets designed for the automotive domain, which we attempt to review in order to identify annotation strategies that can be reused for our use case. Table 1 shows the different datasets proposed in the literature on vision-based risk analysis for autonomous car driving. Among the different criteria that characterize a dataset, two major criteria are analyzed: the annotation framework and the risk assessment metric.

Annotation framework Annotation is a complex task, requiring both time and energy from the annotators. Moreover, decision making algorithms rely on increasingly complex systems that require large datasets with an exhaustive scenario panel. The design of an annotation framework requires some consideration to identify the appropriate strategy providing a fast and efficient tool for annotators, while ensuring high-quality annotations. To reduce the complexity of the annotation process, several techniques are used. Among these techniques, we distinguish two categories. The first category relies on human annotation [START_REF] Wang | Collision Risk Rating of Traffic Scene from Dashboard Cameras[END_REF][START_REF] Corcoran | Traffic Risk Assessment: A Two-Stream Approach Using Dynamic-Attention[END_REF][START_REF] Yurtsever | Risky Action Recognition in Lane Change Video Clips using Deep Spatiotemporal Networks with Segmentation Mask Transfer[END_REF], guaranteeing a high level of analysis while reducing the quantity of annotated data. [START_REF] Wang | Collision Risk Rating of Traffic Scene from Dashboard Cameras[END_REF] assume that accidents are brief compared to other driving situations, so they focus only on short video clips of a few seconds to build their dataset, at the expense of scenario variety. [START_REF] Corcoran | Traffic Risk Assessment: A Two-Stream Approach Using Dynamic-Attention[END_REF] propose to simplify the annotation of a video sequence by applying the same level of risk to the whole clip. However, this technique can lead to misinterpreted risks as it doesn't consider temporal segmentation. The second category relies on automatic techniques to annotate a large set of data very quickly [START_REF] Feth | Dynamic Risk Assessment for Vehicles of Higher Automation Levels by Deep Learning[END_REF], sometimes at the expense of a high level of analysis. [START_REF] Feth | Dynamic Risk Assessment for Vehicles of Higher Automation Levels by Deep Learning[END_REF] use a video game to generate a large dataset on which they apply an automatic metric that provides a 3D simulation-driven measurement. The ideal solution is to balance quality and quantity, but it's complicated due to the limitations of publicly datasets.

In addition to the annotation strategy, it is important to identify the criteria taken into account by the annotators to evaluate a risk. All authors agree that risk annotation is a tricky task and may vary between annotators depending on their interpretation of the scene or different criteria, such as weather, light or context. Besides synthetic datasets, other datasets rely on the expertise of at least three annotators to avoid ambiguity about a situation, although this does not always guarantee a relevant annotation, which

✗ ✓ ✗ ✗ ✗ Weather ✗ ✗ ✗ ✗ ✓ Light ✗ ✗ ✗ ✗ ✓ User ✗ ✗ ✗ ✗ ✓ Collision ✓ ✓ ✓ ✓ ✓
sometimes requires removing data in the initial set [START_REF] Corcoran | Traffic Risk Assessment: A Two-Stream Approach Using Dynamic-Attention[END_REF]. To reduce the large variability in the annotations, the risk is generally categorized in three to five levels, ranging from safe (vehicle at normal speed), to critical risk (unavoidable accident). The granularity of the levels varies between datasets but a very strong overlap exists between all of them. Concerning the annotation criteria, the time to collision is generally taken into consideration by the annotators, either directly estimated by an algorithm [START_REF] Feth | Dynamic Risk Assessment for Vehicles of Higher Automation Levels by Deep Learning[END_REF], or interpreted according to the driver's viewpoint [START_REF] Wang | Collision Risk Rating of Traffic Scene from Dashboard Cameras[END_REF][START_REF] Corcoran | Traffic Risk Assessment: A Two-Stream Approach Using Dynamic-Attention[END_REF][START_REF] Yurtsever | Risky Action Recognition in Lane Change Video Clips using Deep Spatiotemporal Networks with Segmentation Mask Transfer[END_REF]. For this, the analysis of the behavior of other road users is an essential factor that is considered by the annotators to estimate this metric. However, this information is not integrated in the annotations. One dataset is distinguished by the availability of a temporal segmentation of the evolution of the risk [START_REF] Wang | Collision Risk Rating of Traffic Scene from Dashboard Cameras[END_REF].

Risk assessment metric Avoiding accidents is the major task of the decision-making algorithms in autonomous vehicles. Metrics such as time to collision [START_REF] Wang | Collision Risk Rating of Traffic Scene from Dashboard Cameras[END_REF][START_REF] Yurtsever | Risky Action Recognition in Lane Change Video Clips using Deep Spatiotemporal Networks with Segmentation Mask Transfer[END_REF] and time headway [START_REF] Feth | Dynamic Risk Assessment for Vehicles of Higher Automation Levels by Deep Learning[END_REF] are often used to achieve this task. [START_REF] Feth | Dynamic Risk Assessment for Vehicles of Higher Automation Levels by Deep Learning[END_REF] propose a risk metric based on time headway that measures the distance between two vehicles. Although often used, this metric is simplistic and does not take into account the multiple elements of a situation, which often leads to overestimate of risk. Furthermore, it only considers the closest vehicle, which is very limited to a specific scenario, and less suited for the railway domain. [START_REF] Wang | Collision Risk Rating of Traffic Scene from Dashboard Cameras[END_REF] propose a risk metric based on the time to collision. Since accidents are uncommon, they suggest to deal only with cases where accidents occur. The risk annotation is then defined in three levels and time-segmented according to the time to collision. This method is innovative, but their metric is only based on the time to collision, and does not take into account other elements.

To enhance risk metric reliability, [START_REF] Corcoran | Traffic Risk Assessment: A Two-Stream Approach Using Dynamic-Attention[END_REF] incorporates multiple criteria, including factors like the presence of other vehicles, vulnerable road users, traffic flow, and weather, across four risk levels. The authors acknowledge the complexity of annotating various risk factors, leading to situations being annotated at different levels by annotators. In an effort to simplify annotation, a uniform risk level is applied to entire video sequences, encompassing risk-free segments. Although faster to annotate, this method does not clearly identify the risk factors, and induces bias in the training data.

SMART-RD FRAMEWORK

The proposed SMART-RD (riSk assessMent frAmework foR auTonomous Railway Driving) framework is an extension of RailSem19 dataset [START_REF] Zendel | RailSem19: A Dataset for Semantic Rail Scene Understanding[END_REF]. RailSem19 dataset includes 8500 unique images taken from an ego-perspective of a rail vehicle (trains and tramways). Extensive semantic annotations are provided, both geometry-based (track profile) and dense label maps. One contribution of this paper is to use the RailSem19 dataset to produce a new set of annotations corresponding to the risk level in each visual scene viewed from the perspective of the driver.

Annotation tools

Risk annotation involves describing the risk factors that could cause potential damage (hazard identification) and assessing the risk associated with those hazards (risk analysis or risk assessment). Unfortunately, there is no straightforward or unique method for determining the risk level. Determining risk requires knowledge of the activities in the field, the urgency of the situations, and, most importantly, objective judgment. A train operation risk assessment is a thorough examination of various traffic situations to identify objects, situations and processes, that could harm a vulnerable target, and to assess the likelihood and severity of the risk, i.e., to determine the risk level. In our assessment, the risk levels are defined into three categories:

• Secure -visual scenes with no hazard, with a low to no probability of incident (ideal driving situation);

• Moderate -visual scenes involving hazards with a low to medium probability of causing an incident (normal driving situations);

• High -visual scenes with hazards having a high probability of causing an incident (dangerous driving situations).

For each of the RailSem19 dataset images, SMART-RD brought risk annotations over 5 different modalities often observed in the literature: general risk, weather, light, users and context. The choice to extract different risk categories answers the fact that most of works in the literature struggle to assess the risk directly. In the proposed framework, the general risk is divided into the three risk levels aforementioned. The other modalities are broken down into 11 levels from 0 to 10. Fig. 2 illustrates the annotation tool used to annotate the Railsem19 dataset. To avoid annotation bias due to tiredness, the tool makes it possible to divide the dataset into subsets and pick up the annotation process where it was left off.

Annotation guidelines

In order to have a greater statistical significance, the Railsem19 dataset is annotated by three different annotators. It allows to avoid outliers by applying an aggregation method on the three annotations (mean, majority vote, max, etc.) Scenes with a strong disagreement between annotators are also interesting since they probably carry subtle elements that can induce a switch in the risk assessment. They can therefore be a topic of interest by their own. Instead of some work, we voluntarily keep these occurrences in the dataset since they might carry complex but interesting risk features.

Risk assessment is a very subjective task. Therefore, annotators have been given instructions in order to keep a certain level of coherence between their results. Based on a first subset of the data, discussions were held to adjust the evaluation criteria. For each metric, a list of the different elements considered by the annotators during the evaluation was established.

Weather refers to the weather conditions and their implications for the ego-vehicle and other users. Weather is considered dangerous when visibility is reduced (fog) or when it causes changes in track conditions (slippery due to rain or snow). The accumulation of these effects or the severity of the weather conditions also influences the score, such as the fact that snow covers the rail partially or totally.

Light refers to the lighting condition in the scene. The risk score varies depending on the amount of light, but also on the contrast, for example at sunrise and sunset. In some situations, light rays strongly reduce the visibility of the sensors, which makes decision making extremely difficult. Users refers to all other dynamic users of the scene. The risk factor is mainly based on collision between the vehicle and the user. Each obstacle is a potential risk, whatever its nature (object, animal or person). Several criteria are considered to evaluate the level of danger that the obstacle represents. Size, estimated speed, trajectory, distance from the vehicle and from the lane, and density are criteria to consider. When it comes to living obstacles (animals or people), behavioral analysis is also an essential criterion, including features like posture, gaze or attention in order to anticipate the reaction of this user to the train passage.

Context refers to the environment in which the train is located. The risk factor is mainly related to the conditions surrounding the train, such as the presence of rail/road crossing and whether they are guarded or not. It also depends on certain situations, such as entering/leaving a station, passing through a tunnel, etc.

The infrastructures around the railroad can also cause occlusions. The risk is even higher if the track is not protected by barriers or if the train arrives at the beginning of a curve, in which case, the sensors do not cover the same field.

It is apparent that the annotation does not contain all these details. However, having all the criteria used to evaluate the situations, it is easier to design decision making algorithms to match the estimated scores on each metric. Fig. 3 illustrates an example of annotation performed on three images each corresponding to one of the three general risk levels: secure, moderate and high.

Annotation correlation

In order to study inter-rater agreement, several statistical measures were performed: the Cronbach's α measures, the Intraclass Correlation Coefficient (ICC) and the Krippendorff's α measures. These measures were applied to each modality to identify correlations within the same metric and between different metrics. Cronbach's α measures if a group of items (the annotators) consistently attributes scores to another set of subjects (the images). Its definition is the following one:

α = k k -1 (1 - ∑ k n=1 σ 2 Y n σ 2 X ) (1)
where σ 2 Y n is the variance of the scores of n th item, σ 2 X the total variance of the scores and k the number of items, i.e. 3 in our case. A value of 0.8 is considered [START_REF] Nunnally | Psychometric theory / Jum C. Nunnally[END_REF] as a good threshold for the Cronbach's α. The ICC scores are obtained through an ANOVA-based framework using the pysch R module.

Similarly to the Cronbach's α, the ICC ranges from 0 to 1. Interpretation guidelines [START_REF] Cicchetti | Guidelines, criteria, and rules of thumb for evaluating normed and standardized assessment instrument in psychology[END_REF] suggest fair agreement between 0.4 and 0.59, good agreement between 0.6 and 0.74, and excellent agreement above. Finally the Krippendorff's α measures the ration between the actual disagreement and the expected disagreement by chance, ranging from -1 to 1. Negative values means a complete disagreement and 1 a perfect agreement between raters. In general, values larger than 0.667 can be considered to draw conclusions.

From the scales given above and the results obtained in Table 2, the three annotators share mainly a same consistency in their annotations. The correlation scores reveal two groups within the modalities: strong correlations among annotators for "weather", "light" and "users" and less consistency for "general" and "context". The common characteristic between the modalities of the first group is is their objectivity and explicit features. The risk of weather or light leaves very little room for personal interpretation. Similarly, the users risk mainly depends on the number of users, their distances to the train, their positions with respect to the tracks or their body orientations which are all factual and explicit features. The context modality is more inconsistent since it carries subjectivity and requires an in-depth analysis of the content in the scene. This may explain why there is very few work in the literature on contextual risk analysis. Since the general risk strongly depends on the context, i.e. annotators classify very few rural scenes as risky, the general risk also perceives this decrease in consistency.

To further investigate the annotations provided by the panel of annotators, it is interesting to consider the correlations between the general risk score associated with the situation, with respect to the different criteria: weather, light, users and context. This analysis is based on the Pearson score which can be computed for two samples of random variables with the following formula:

ρ x,y = n ∑ x i y i -∑ x i ∑ y i n ∑ x 2 i -(∑ x i ) 2 n ∑ y 2 i -(∑ y i ) 2 (2)
where x i and y i are sample points of the two respective random variables and n the size of both samples. From the graphs, the "users" and "context" modalities are much more correlated with general risk. Indeed, when the annotators put high values of risk for these two modalities, the general risk is most of the time set to "High" represented by the value 2 on the graph. Annotators often qualify the general risk as a collision risk and therefore give more weight to these two modalities. The Pearson correlation coefficient of the "users" and "context" modalities with respect to the general risk are 0.60 and 0.61 respectively which can be considered as high values. There is also a strong correlation (0.58) between the two modalities themselves since a risky context is often considered as a place where it is very likely to meet a user. However, this obviously does not imply that these modalities are interchangeable. Indeed, a highly urbanized area with no pedestrians cannot always be considered as safe as they may appear from obstructed areas. The "weather" and "light" modalities can be rather seen as exacerbating factors rather than risk causing factors. Indeed, Fig. 4 also shows that the highest level of risk for these modalities does not imply the highest level of general risk. The correlation for low values with the general risk is also very weak, i.e. annotators only take these modalities into account for the general risk when they reach relatively high values. This is again reflected in the Pearson correlation coefficient of 0.23 and 0.27 for the "weather" and "light" modalities respectively.

EXPERIMENTS

In this section, we propose several studies for the risk assessment using the proposed benchmark. Using the RGB images of the RailSem19 dataset [START_REF] Zendel | RailSem19: A Dataset for Semantic Rail Scene Understanding[END_REF] as input of a neural network, we perform a binary risk assessment: classification between secure and risky based on the different data modalities, different models and different data aggregation methods.

Evaluation protocol

Validation protocol In order to evaluate the decision making model and to check the good performance of the model when faced with unknown data, a stratified k-fold cross validation protocol is used, with k = 3. The data is partitioned into training and testing sets following an 80/20 distribution. The reported accuracy in the subsequent tables is derived from a balanced subset of the test set. Additionally, for various evaluations, the performances represent the average accuracy calculated across the three folds, along with the corresponding standard deviation.

Data imbalanced In risk analysis datasets, highrisk situations are generally under-represented, leading to data imbalance. The same applies to the proposed dataset, as illustrated in Fig. 5. First, to maintain the spatial coherence of the elements in the scene, e.i., image centered on the rail and environment on the sides, only a horizontal flip augmentation is applied. Poor results have been observed by applying augmentations on light changes or by adding noise (blur, distortion) to the images. Also, only a balanced sample data was selected in the training and test tasks since binary classes are very imbalanced and irregular between risk modalities.

Implementation Details

Two different models have been tested in this study : a CNN, and a Vision Transformer (ViT) [START_REF] Dosovitskiy | An image is worth 16x16 words: Transformers for image recognition at scale[END_REF]. The ViT has been pretrained on ImageNet-21k [START_REF] Deng | Imagenet: A large-scale hierarchical image database[END_REF]. The original clas-sifier head has been removed which is the fully connected one to replace it by a new one which fits our binary classification problem. We only train this new classifier since experiments have shown that unfreezing the last convolutional layer does not provided significant improvements. We implemented the model in PyTorch and conducted experiments on a single GeForce RTX 3060 GPU with 12GB VRAM. We employed the AdamW optimizer [START_REF] Loshchilov | Decoupled weight decay regularization[END_REF] with fixed learning rates of 0.01. The images are resized to a 224x224 shape and normalized using the mean and standard deviation of the ImageNet dataset [START_REF] Deng | Imagenet: A large-scale hierarchical image database[END_REF]. The batch size chosen is of size 32. Each model are trained for 30 epochs.

Quantitative experiment

A first baseline based on a binary danger or nondanger classification and the median aggregation function for labeling the risk level according to the 3 annotations is proposed in Table 3. We compare the performance of the CNN model and ViT model pretrained on ImageNET on the different modalities selected by the annotator panel: general risk (general), weather conditions, visibility (light), presence of vulnerable road users (users) and driving context related to the environment (context).

Considering the performances, the pre-trained ViT model gives the best performance, whatever the modality observed, showcasing the effectiveness of the ImageNet-based pre-training for this task. Given the results obtained on the different modalities, risk estimates based on visibility are well-identified for both models, while results for other modalities exhibit significant variations between models. The performance of the pre-trained ViT model underscores the potential of attention-based architectures in risk analysis.

To investigate the impact of subjective interpretation between annotators in model learning, a study of aggregation functions is carried out in Table 4. The functions correspond to the highest level of risk reported by the 3 annotators (Max), the lowest (Min) and the median (Median). For this study, only the pre-trained Vit model is used, given its better per- formance. Overall, considering the median score gives better performance. With regard to the different modalities, user-based risk is the most controversial between the Min and Max annotations. Two observations may explain this result. Firstly, the accident involving a human factor has a more serious denotation from the driver's point of view. In this case, one of the annotators considered that a car crossing the path of the train was less risky than a pedestrian. Secondly, few variations in the annotations were observed depending on the distance or position of the users in relation to the train. Indeed, one of the annotators considered that a pedestrian waiting on the platform did not represent a serious danger, whereas the other annotators tended to put a high level of danger as soon as the train was at a certain distance from the pedestrian, regardless of the context. Human interpretation may have a slight impact on risk annotation, particularly on the users and context modalities, so a study presented in Table 5 is proposed to determine which class separation strategy enhances the model's capacity to distinguish between risky and not-risky situations. Several strategies have been proposed, in the following format (non-risk score categories, risk score categories) : S1 {(0),(1-10)} ; S2 {(0),(6-10)} ; S3 {(0-2),(3-10)} and S4 {(0-5),(6-10)}. In view of the performances, the S2 strategy is the most suitable for risk discrimination between the different modalities. By excluding low scores, it's easier to separate the R and R classes. Small scores are often more open to human interpretation, and tend to vary between different users, particularly with regard to user-based risk. We propose the first framework on risk analysis for autonomous railway driving, named SMART-RD.

Based on the RailSem19 dataset, SMART-RD provides additional annotations on the general risk based on weather, light, users and context. A first study is performed on these data according to a binary danger or non-danger classification. The proposed baseline is based on traditional learning models in image processing and does not take into account recent advances in the literature for risk analysis. In future work, we explore more complex decision models better adapted to risk assessment, taking into account the per-modality annotation to assess the general risk.

Figure 1 :

 1 Figure 1: Overview of the SMART-RD framework.

Figure 2 :

 2 Figure 2: Risk annotation tools enabling to browse the Railsem19 dataset and to associate a risk level based on 5 criteria: general risk, weather, light, users and context according to 2 different scales.

Figure 3 :

 3 Figure 3: Proposed annotations in SMART-RD.

Figure 4 :

 4 Figure 4: Mean general risk level for each value of the different criteria: weather, light, users and context.

Figure 5 :

 5 Figure 5: Distribution of data provided in SMART-RD according to each modality. Distribution histograms are enhanced by three images extracted from the dataset, representing scores of 0, 5 and 10 respectively.

Table 1 :

 1

		Vision-based risk analysis dataset for autonomous
	driving. References: A-(Feth et al., 2018); B-(Wang
	and Kato, 2017); C-(Corcoran and Clark, 2019) and D-
	(Yurtsever et al., 2019).
		References	A	B	C	D	Our
	Settings	Real Nb Data Type Availability	✗ 110k images video ✓ 1,890 ✗ ✗	✓ 1,750 video ✗	✓ 860 video ✗	✓ 8,500 images ✓
		Labels	score 3 levels 4 levels 5 levels 3 levels
	Annotations	Nb Anno. Temporal	auto	3 + 1	3	10	3

Table 2 :

 2 Different correlation scores applied on the annotations made by the panel of annotators according to the 5 criteria to evaluate the risk. (↑) The closer the score is to 1, the stronger the correlation is.

	Metric	General Weather Light Users Context
	Kripp. α ↑	0.59	0.72	0.74 0.78	0.68
	ICC(3,1) ↑	0.60	0.74	0.75 0.78	0.69
	Cronbach's α ↑	0.82	0.89	0.90 0.91	0.85

Table 3 :

 3 Comparison of the performance of learning models on the binary classification task on different modalities involving a risk for driving.

	Methods General Weather	Light	Users	Context
	CNN	65,67% 64,67% 73,67% 68,67% 69,00% ±2, 08 ±2, 08 ±1, 53 ±8, 39 ±5, 20
	ViT pre.	77,67% 76,00% 75,33% 81,33% 76,00% ±2, 08 ±2, 65 ±7, 37 ±3, 51 ±1, 00

Table 4 :

 4 Comparison of the data aggregation functions used with the pretrained vision transformers.

	Aggregation General Weather	Light	Users	Context
	Maximum	77,00% 75,33% 74,00% 79,67% 74,33% ±0, 00 ±4, 16 ±3, 00 ±3, 21 ±1, 53
	Minimum	72,33% 69,67% 75,67% 66,00% 79,33% ±3, 21 ±3, 79 ±3, 06 ±3, 61 ±8, 33
	Median	77,67% 76,00% 75,33% 81,33% 76,00% ±2, 08 ±2, 65 ±7, 37 ±3, 51 ±1, 00

Table 5 :

 5 Comparison of the binary class (risky (R) and nonrisky (R)) separation methods used with the pretrained ViT.

	Strategy Separation Weather	Light	Users	Context
	S1	R:(0) R:(1-10)	70,67% 87,00% 81,67% 77,33% ±6, 66 ±3, 00 ±5, 86 ±4, 51
	S2	R:(0) R:(6-10)	86,33% 94,33% 80,67% 88,67% ±4, 51 ±1, 53 ±7, 51 ±5, 03
	S3	R:(0-2) R:(3-10)	80,33% 86,00% 66,67% 80,00% ±4, 51 ±1, 00 ±5, 03 ±2, 65
	S4	R:(0-5) R:(6-10)	77,00% 93,33% 77,67% 77,00% ±10, 44 ±2, 31 ±6, 11 ±4, 58

ACKNOWLEDGEMENTS

This project was supported by the France 2030 Investment Plan operated by ADEME.


Enzo Isnard

Sébastien Héron

Stéphane Lanteri

Mahmoud Elsawy

Advancing wavefront shaping with resonant metasurface

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

Advancing wavefront shaping with resonant metasurface

Enzo Isnard * ,1,2 , Sébastien Héron 2 , Stéphane Lanteri 1 , Mahmoud Elsawy * ,1

1 Université Côte d'Azur, Inria, CNRS, LJAD, 06902 Sophia Antipolis Cedex, France 2 THALES Research & Technology, 1 avenue Augustin Fresnel, 91120 Palaiseau, France enzo.isnard@thalesgroup.com mahmoud.elsawy@inria.fr

In metasurface design, the use of look-up tables based on a local periodicity approximation have been traditionally employed, but can result in sub-optimal designs due to lack of consideration of near-field coupling effects, which are particularly important for resonant systems. This paper explores the benefits of taking into account near-field coupling while optimizing non-local resonant metasurfaces to enhance their performance for wavefront shaping, including the state-of-the-art Huygens's metasurface.

Summary

Fig. 1. Deflection efficiency of metagratings obtained with a look-up table and with global optimization. The inset indicates the schematic representation of the structure under consideration [START_REF] Özdemir | Polarization independent high transmission large numerical aperture laser beam focusing and deflection by dielectric huygens' metasurfaces[END_REF] Metasurfaces are two-dimensional planar structures constituted of sub-wavelength sized elements that can alter electromagnetic wave properties, including phase, amplitude, and polarization. Metasurfaces are generally locally approximated by periodic structures around each nano-element. This approximation allows the use of the look-up tables to design them, consisting in simulating the optical response of isolated nano-elements and placing them according to a phase profile given by the generalized Snell's law. In highly resonant structures the meta-atoms are not isolated and their response may depend on their neighbors, making the local periodicity approximation irrelevant. In our previous work, we exploited the benefits of global optimization coupled with full-wave solver to significantly improve the performances of metasurfaces [START_REF] Elsawy | Global optimization of metasurface designs using statistical learning methods[END_REF][START_REF] Elsawy | Multiobjective statistical learning optimization of rgb metalens[END_REF]. The present work considers resonant configurations including the state-of-the-art Huygens's metasurfaces where the apparition of collective resonances [START_REF] Gigli | Fundamental limitations of huygens' metasurfaces for optical beam shaping[END_REF] makes the local periodicity assumption obsolete. We use Kriging-based Bayesian optimization as it is well suited for expansive black-box function and can be adapted to handle fabrication uncertainty [START_REF] Elsawy | Optimization of metasurfaces under geometrical uncertainty using statistical learning[END_REF] and multi-objective problems [START_REF] Elsawy | Multiobjective statistical learning optimization of rgb metalens[END_REF]. We illustrate our methodology in the case of a meta-deflector taken from ref. [START_REF] Özdemir | Polarization independent high transmission large numerical aperture laser beam focusing and deflection by dielectric huygens' metasurfaces[END_REF]. Figure 1 shows the deflection efficiency of both the optimized and the look-up table design, where simulations are performed using the software suite DIOGENeS [START_REF]DIOGENeS : A Discontinuous-Galerkin based software suite for nano-optics[END_REF] developed at Inria Sophia Antipolis. The drastic improvement of efficiency highlights the need for optimizing the whole period at once in the case of highly resonant structures.


Antoine Henry
email: antoine.henry@univ-lille.fr

Alain Mille
email: alain.mille@univ-lyon1.fr

Médard Agbayazon

The building of pedagogical material, an alternative way to educate using educational commons

The building of pedagogical material, an alternative way

The building of pedagogical material, an alternative way to educate using educational commons

Abstract: Based on the Je Fabrique mon matériel pédagogique project, this article aims to explore a way for teachers and children to develop pedagogical material through the lens of the commons approach. Beyond market-driven logic, in this article, we will explore two dimensions, appropriation and articulation, to explore this perspective as a way to think about education.

Introduction

This communication is based on the case of the Je Fabrique mon matériel pédagogique project1 within Francophonie. The aim of this project is to help teachers easily create the pedagogical material that they need or building materials that have already been made by someone else beyond market-driven logic. Indeed, understanding educational resources as common resources is an opportunity to give everyone the means to do, to experiment but also to be in a mutual aid logic by going beyond the competitive thinking of the market. If thinking education in common is perceived as a condition in a democratic and inclusive perspective (De Lissovoy 2011), a direct link has been made to think education as a common [START_REF] Fee | Faire école en Commun(s). L'école sans école : Ce que le confinement nous dit de Caen[END_REF] or to think about education and commons [START_REF] Korsgaard | Education and the concept of commons. A pedagogical reinterpretation[END_REF]Means Ford & Slater 2017).

The project also finds its roots in the maker spirit (i.e., the Maker Faire2 , a gathering of makers and educators). The "Maker movement" emerged in the 2000s in the United States under the impetus of Dale Dougherty to bring together heterogeneous communities of people practising personal fabrication in various ways under a single name [START_REF] Genatio | Des «makers» en «mouvement»? Sur l'origine, l'utilisation et la pertinence de la notion de « Mouvement Maker[END_REF]. In this project, we do focus on pedagogical material; we mobilize contributions relating to peer production [START_REF] Benkler | Commons-based Peer Production and Virtue[END_REF][START_REF] Benkler | Peer Production: A form of collective intelligence[END_REF], which they associate with a form of collective intelligence, with the commons-based peer production [START_REF] Kostakis | The convergence of digital commons with local manufacturing from a degrowth perspective: Two illustrative cases[END_REF][START_REF] Kostakis | Commons-based peer production and digital fabrication: The case of a RepRap-based, Lego-built 3D printing-milling machine[END_REF] and distributed manufacturing (ibid. )3 .

In this project, we are over two important dimensions:

-appropriation, in a sociotechnical meaning, the question of appropriation involves the inclusion of the object (here an educational common) in an artifactual process that begins with the discovery of the object studied, its functionalities, and its possibilities, before moving on to an eventual assimilation of the object into the operator's daily life and then its appropriation. Once this stage is reached, the object can even become invisible in its context of use [START_REF] Rabardel | Instrument, activité et développement du pouvoir d'agir[END_REF]. We would like to study the conditions of a wide appropriation of resources with a concern for cross-validation bringing together an academic community and civil society. -And of articulation, here, how the educational common 'fits' into the professional activity and the culture of teachers, children or even parents to become established. Indeed, commons in education could animate attempts to transform the substance of our relationship to teaching, learning, research, and institutions of education in accordance with the spirit of the commons. Education would be transfigured, then, into a collective good, which is created, governed, and enjoyed in common by all parties of the educational community. In this way, we can envision an education organized "as an institution of the commons" such that the management of knowledge and education will be a collective process and educational communities will organize and coordinate among themselves on the basis of the "democratic participation process". Such a learning community should rely on the values of contingency, experimentation, and surprise in the sense that when learning is coproduced and comanaged by all members of the community, its trajectory cannot be developed in a linear and predefined way from one stage to the other; therefore, openness to the creativity of the community and trust in an outcome that cannot be fully anticipated is a prerequisite [START_REF] Pechtelidis | Education as Commons, Children as Commoners: The Case Study of the Little Tree Community[END_REF].

In this way, we are moving from a conception of the school centred on itself to a school open to the heart of the city from the perspective of "learning territory", which is "a place, a time and a network where learning is encouraged in situ and in vivo, outside the walls of institutions, by associating with other actors in the environment. It is a situational process in which sharing knowledge through doing becomes a fundamental function for deploying an imagination, creating visions and participatory policies for the cross-development of individuals, organizations and territories and the well-being of all" [START_REF] Gwiazdzinski | Territoires Apprenants. Seyssinet-Pariset[END_REF]. Open to its territory, inclusive, the school forms citizens and becomes a place of concrete expression of democracy.

Specific material for specifics contexts and children

For the teachers, by constructing teaching materials designed as a common resource, it is then possible for them to reproduce it and, more particularly, to adapt it to the context in which they are working. For example, several teachers said that they did not necessarily have electricity or, outside the capital, even a lack of resources that could be used to create pedagogical material. The framework of the commons is therefore conducive to the appropriation and, above all, the adaptation of this resource to the contexts in which teachers work. The material can be built using the resources available to the teachers. This is essential if the context in which they work is to be considered.

Several other dimensions emerged from the interviews. Our interviews highlight 1) Teachers should involve children in the classroom and consider their way of learning.

By creating the material, for teachers, it's a means of "going to the real world" (Cirima, physique teacher). Indeed, the question of materialization comes up regularly in the interviews. This element is stimulating for teachers, as they feel proud of themselves: "The fact that I can recycle objects at hand, make a teaching tool to make it easier for a learner to understand, that fascinates me. It opens my mind to many other things" (Cirima, physique teacher). In addition, some teachers point out that this can be a way for them to gain more respect from their pupils and to answer one of their issues: "we do not have enough materials, apart from textbooks and notebooks" (Dominique, Spanish teacher).

Regarding the children, one teacher highlights that he would like to make them create their own material because, for him: "it's a way of highlighting children's talents, working on their openness" (Yao, teacher). In addition, it is stimulating for teachers, as each year will be different, as they, or the children, can make different materials according to the context and their will (Kouakou, history and geography teacher).

2) The willingness of makers/teachers to create another environment (i.e., in a FabLab) where children (and even adults) with disabilities or difficulties at school can learn by doing, and this way regain confidence.

This approach is perceived as an experimental and concrete approach for teachers to:

• Create a space other than the institutional space to offer students other ways of doing things ("to the realities of my learners"). Moreover, by working with FabLab, they can do school outside of the school or in relation to their territory. • An aid for orientation, indeed, this approach can be viewed as an alternative way to include people who do not have the dominant values of school. • Interest in escaping the monotony of practice. Indeed, building the material is interesting, it allows you to observe it, to appropriate it and to think about how to transform it. • It's an interesting way to think something specific for children with disabilities (see Figure 1 after). Indeed, this question of disabilities was important for teachers, as there is no specific support system for them. Therefore, during the workshop, two projects, out of six, were dedicated to children with disabilities. Nearly 1/3 of the interviewees mentioned that this approach can help them this kind of pupil. In this way, it is possible to create something specifically rooted in the disability of the child.

To illustrate this dimension, Figure 1 represents a specific material created to let hearingimpaired children easily catch the attention of the teacher to say if he/she is following or not. Teaching material rooted in a sociotechnical context [START_REF] Lauwerier | Éducation non formelle, alphabétisation et communautés locales en Afrique de l'Ouest francophone[END_REF] emphasized the importance of local communities in the provision of education for illiterate adults and out-of-school children in the context of francophone West Africa. Therefore, to strengthen the learning network and equip education actors with resources that can be used in their context, we find this logic of anchoring education in the city, communities and networks. Here, pedagogical situations are described in light of the mobilized resources and networks (for instance, the ReFFAO (Francophone Fablab Network of West Africa))5 . Both in terms of creating material adapted to the technical environment (resources, equipment, etc.) and to the context of the children. This element resonates with the important culture of resourcefulness in the country.

Teachers from the workshop had an important willingness to contribute at a larger level than individuals and to do something that can be helpful/adaptable for other teachers:

• Related to their constraints.

• The important number of children in class. How can they learn in a different way and, moreover, for children with disabilities? • Regardless of the subject taught. For instance, language teachers (French, Spanish and English) were part of a project to help children learn language, and they easily saw how to adapt it to their own context.

From the sociotechnical perspective, we need to take into consideration appropriation and articulation to take into consideration their contexts (political, economic and social), their means and their needs to pursue their educational purpose.

Regarding appropriation, during this event, teachers were in a Fablab6 to help them think and realize a pedagogical tool that is rooted in their context and needs. In this approach, the discovery of the new artifact (here a pedagogical tool) occurred while the artifact was designed and then built (live instrumental genesis). It can then be experimented in the situation and improved through feedback. In this way, the output is depositary of their share representation system and experience in their teaching activity. Considering that the development of the pedagogical material is linked to its use, it finds itself in a situation that can be described as incomplete by design [START_REF] Garud | Incomplete by Design and Designing for Incompleteness[END_REF]. It remains incomplete from the point of view of uses, which remain fluid and unstable; this means paying close attention to actual, current uses, rather than potential or imagined ones. [START_REF] Folcher | Conception pour et dans l'usage : La maîtrise d'usage en conduite de projet[END_REF] refers to creators as "designers in use". This design in the use allows easing the articulation with the current practice, i.e., Elodie (teacher of earth and life sciences) would like to print a 3D flower cut in half that is not withering.

This flexibility is allowed by an intrinsic dimension of the commons, which is a free resource that can be modified by anyone. Moreover, if someone transforms a resource (for example, to be used by blind people), then it is possible to share it broadly.

Limitations and questions

With this first work, we need to take into consideration some limitations and questions that have arisen.

1) Problems of scaling up (lack of infrastructure, capacity to set up the same approach on a whole territory, volunteers, etc.), what support(s)? Indeed, the commons approach has an interesting link with the local dimension. However, the question arises of scaling up a local educational common so that it can be disseminated and reappropriated in other contexts.

2) The question of trust. Indeed, some answers are based on trust. Trust is essential for bringing together heterogeneous groups [START_REF] Wenger | Cultivating communities of practice[END_REF] but also for encouraging them to share knowledge, resources and practices. In particular, this trust requires members to develop a sense of belonging to the community. 3) What measure(s) should be used to analyse the effects of this approach? 4) What validity for the resources produced? Governance? 5) This project makes an important link between FabLabs and schools. However, African FabLabs remain fragile and highly dependent on two elements (Liotard 2020): a. the energy and personality of the fabmanager on the one hand; b. and international aid in many forms to support these places. 6) As educational commons are part of knowledge commons, we need to take into consideration how they are organized. A specific focus can be placed on the input for knowledge organization (KO) and ethical questions related to KO.

Discussion and Conclusion

This project leads us to think about communities through the lens of education and commons. Indeed, the community can begin to live in common, with reciprocity that values the work of creation as well as the work of use. It is the people who value the contents of these platforms by making it possible to put their uses into a narrative and to more easily identify how to mobilize resources in a specific situation. In addition, it's also them that are going to help the community to survive.

We can work over the combination of organizational, community and individual levels to allow us to reach an understanding of the educational context situated in practices and usages from the individual to the global level. There, we can make the link with collective intelligence and the capacity to the people to change institutions or organizations through the deployment of their thought patterns [START_REF] Henry | Platform and Collective Intelligence[END_REF].

The creation of pedagogical materials directly by teachers or students makes a bridge between educational commons and the maker movement. Using this approach allows working over two important dimensions:

-appropriation to ensure that the material is grounded from the context and needs related to its use. In addition, as the environment/needs evolve, the materials can also be updated to take into consideration this new situation or by transformation by someone else to another context; -and articulation to facilitate the fitting of the material to the environment (i.e., low resources) and within the culture of people.

According to the context, like teachers of Ivory Coast, this approach could help teachers and children (for instance, with specific disabilities) by fitting within the constraints that we can encounter. Children can also be part of the teaching by contributing, or even creating, to the production of their materials. To go further, thinking pedagogical material as commons is a way to allow all the stakeholders related to education to be a concrete part of it and build a more inclusive and democratic school.

Figure 1 A

 1 Figure 1 A materiel created to let hearing-impaired children easily catch the attention of the teacher. Created by Koffi Eulodie, Diakabia Kone, Kouassi Ettie J.P., Ouroubie Pascal Eric, Saliou Nassirou, Marie Auxiliatrice Da Silveira

  Between the 29 th of October 2022 and the 5 th of November 2022, a series of interviews were conducted in Abidjan (Ivory Coast) during the MakeAfrica conference 4 to animate a dedicated workshop with thirty teachers from all the country and with makers from other African countries. Through interviews from teachers and makers and with examples of what was made during this week, we performed research. In this work, we explore what educational commons are and how educational commons can be a way to answer day-to-day needs from teachers and students in the Ivory Coast and contribute to transforming education.

https://fabriqueedu.tierslieuxedu.org/

https://makerfaire.com/

Distributed manufacturing is a system in which the production of goods is carried out in geographically dispersed micromanufacturing units linked together by telecommunication tools. This system is now seen as a credible possibility, thanks in particular to the development of the Internet on the one hand, and to the fall in the price of numerically controlled machines on the other, which has led some authors to speak of the 'third industrial revolution'[START_REF] Anderson | Makers : La nouvelle révolution industrielle[END_REF][START_REF] Rifkin | La nouvelle société à coût marginal zéro[END_REF].

https://makeafrica.net/

https://reffao.org/

As they can need some expertise in creation, design (with specific software, i.e., Inkscape) or building (i.e., 3D printing)


Elisa Thoral
email: elisa.thoral@biol.lu.se

Lauréliane Dargère

Ione Medina-Suárez

Angéline Clair

Laetitia Averty

Justine Sigaud

Anne Morales

Karine Salin
email: karine.salin@ifremer.fr

Loïc Teulier
email: loic.teulier@univ-lyon1.fr

Thoral Elisa

Dargère Lauréliane

Medina-Suárez Ione

Clair Angéline

Averty Laetitia

Sigaud Justine

Salin Karine

Teulier Loïc

Non-lethal sampling for assessment of mitochondrial function does not affect metabolic rate and swimming performance

Keywords: red muscle, whole-animal performance, repeatability, goldfish

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

Introduction

Metabolic rate determines the energy expenditure of individual animals and, as such, is a fundamental trait underlying organismal performance [START_REF] Metcalfe | Does individual variation in metabolic phenotype predict fish behaviour and performance?[END_REF]. Metabolic rate is often determined from the organismal rate of oxygen consumption; it is refereed as minimal, or standard, and maximal metabolic rates (SMR and MMR, respectively) in ectotherms. SMR is the minimum of energy that animals expend on their tissues maintenance and homeostatic mechanisms [START_REF] Fry | The effect of environmental factors on the physiology of fish[END_REF]. The MMR is the excess energy an individual can allocate to growth, reproduction [START_REF] Norin | Measurement and relevance of maximum metabolic rate in fishes[END_REF] or exercise [START_REF] Mckechnie | Sources and significance of variation in basal, summit and maximal metabolic rates in birds[END_REF]Weibel and Hoppeler, 2005). Therefore, SMR and MMR represent lower and upper limits of aerobic capacity. However, oxygen consumption alone is unlikely to be a sufficient marker of aerobic metabolism in many situations. This is due to the variability in the link between the amount of adenosine triphosphate (ATP) generated per molecule of oxygen consumed by mitochondria.

Because ATP is the main form of energy fueling cellular function, such as biomass production and muscle contraction, the efficacy with which mitochondria produce ATP can be a major determinant of organismal performance [START_REF] Hood | The mitochondrial contribution to animal performance, adaptation, and life-history variation[END_REF][START_REF] Koch | Integrating Mitochondrial Aerobic Metabolism into Ecology and Evolution[END_REF].

Investigating variation in mitochondrial metabolism provides an opportunity for integrating how mechanisms at the cellular level constraints whole-organism traits [START_REF] Cominassi | Metabolic rate increases with acclimation temperature and is associated with mitochondrial function in some tissues of threespine stickleback[END_REF][START_REF] Conley | Mitochondria to motion: Optimizing oxidative phosphorylation to improve exercise performance[END_REF][START_REF] Hood | The mitochondrial contribution to animal performance, adaptation, and life-history variation[END_REF][START_REF] Koch | Integrating Mitochondrial Aerobic Metabolism into Ecology and Evolution[END_REF]. For example, individual variation in SMR and MMR were positively associated with proton leak respiration in hepatic mitochondria (i.e., LEAK respiration) of brown trout Salmo trutta [START_REF] Salin | Variation in metabolic rate among individuals is related to tissue-specific differences in mitochondrial leak respiration[END_REF]. These results suggest that a high SMR or MMR, rather than signalling a higher ability for respiration-driven ATP synthesis, may actually reflect a greater dissipation of energy. Conversely, the basal metabolic rate in capped chickadees Poecile atricapillus was positively correlated with the phosphorylating respiration (i.e., OXPHOS respiration) in liver mitochondria [START_REF] Milbergue | How does mitochondrial function relate to thermogenic capacity and basal metabolic rate in small birds ?[END_REF]. Similarly, high rates of OXPHOS respiration in heart mitochondria was found in individuals Gulf killifish Fundulus grandis that had the highest MMR (Rees et al., 2022), supporting the idea that mitochondrial properties determine the whole-organism metabolism. Individuals that have higher metabolic rates -and so higher mitochondrial phosphorylating respiration -may therefore have a higher rate of ATP production and perform better. However, the interpretation of cross-sectional correlational studies is limited since any relationship between the mitochondrial and whole-animal performance cannot be presumed causal. The main reason for results derived from correlative approaches is that studying mitochondrial metabolic traits usually involves terminal sampling, where determination of whole-organism performance can only precede the mitochondrial measurement.

There is thus a real need for studies that assess mitochondria from samples that can be collected without sacrificing animals [START_REF] Salin | Variation in the link between oxygen consumption and ATP production, and its relevance for animal performance[END_REF][START_REF] Stier | How to measure mitochondrial function in birds using red blood cells: a case study in the king penguin and perspectives in ecology and evolution[END_REF]. Recently, researchers have sought to conduct non-lethal studies of mitochondrial function [START_REF] Jacobs | Twenty-eight days at 3454-m altitude diminishes respiratory capacity but enhances efficiency in human skeletal muscle mitochondria[END_REF][START_REF] Quéméneur | The relationships between growth rate and mitochondrial metabolism varies over time[END_REF][START_REF] Stier | How to measure mitochondrial function in birds using red blood cells: a case study in the king penguin and perspectives in ecology and evolution[END_REF]. Such new approaches, based on biopsy [START_REF] Pesta | Chapter 3 -High-Resolution Respirometry: OXPHOS Protocols for Human Cells and Permeabilized Fibers from Small Biopsies of Human Muscle[END_REF][START_REF] Quéméneur | The relationships between growth rate and mitochondrial metabolism varies over time[END_REF][START_REF] Teulier | Selective upregulation of lipid metabolism in skeletal muscle of foraging juvenile king penguins: An integrative study[END_REF] or blood samples [START_REF] Amir Dache | Blood contains circulating cell-free respiratory competent mitochondria[END_REF][START_REF] Braganza | Blood-based bioenergetics: An emerging translational and clinical tool[END_REF][START_REF] Fang | Morphological and cytochemical studies of peripheral blood cells of Schizothorax prenanti[END_REF][START_REF] Karamercan | Can peripheral blood mononuclear cells be used as a proxy for mitochondrial dysfunction in vital organs during hemorrhagic shock and resuscitation?[END_REF][START_REF] Stier | How to measure mitochondrial function in birds using red blood cells: a case study in the king penguin and perspectives in ecology and evolution[END_REF], now make it possible to gather information on the temporal dynamics of whole-organism and mitochondrial traits. In birds, non-lethal sampling of blood has shown a relationship between mitochondrial traits in red blood cells and reproductive performance [START_REF] Stier | How to measure mitochondrial function in birds using red blood cells: a case study in the king penguin and perspectives in ecology and evolution[END_REF][START_REF] Stier | Experimental demonstration of prenatal programming of mitochondrial aerobic metabolism lasting until adulthood[END_REF] and whole-animal metabolic rate [START_REF] Malkoc | Inferring Whole-Organism Metabolic Rate From Red Blood Cells in Birds[END_REF]Thoral et al., submitted). In European Sea bass Dicentrarchus labrax, non-lethal sampling of the red muscle has linked variation in mitochondrial properties and individual growth performance [START_REF] Quéméneur | The relationships between growth rate and mitochondrial metabolism varies over time[END_REF]. However, in this last study, the mitochondrial traits that explained growth variation differed between the growth rates determined before and after the mitochondrial assay: past growth was correlated with the activity of the cytochrome c oxidase, a measure of mitochondrial density, whereas future growth was linked to LEAK respiration. A possible explanation is that mitochondrial metabolism varies over time [START_REF] Bouchard | Time course of the response of mitochondria from oxidative muscle during thermal acclimation of rainbow trout, Oncorhynchus mykiss[END_REF][START_REF] Boutilier | Adaptive plasticity of skeletal muscle energetics in hibernating frogs: Mitochondrial proton leak during metabolic depression[END_REF][START_REF] Salmón | Mitochondrial function declines with age within individuals but is not linked to the pattern of growth or mortality risk in zebra finch[END_REF]. This might be representative of natural variation in mitochondrial metabolic traits or because sampling procedure for mitochondrial assay have consequences for upcoming whole-organism performance. It has been shown that muscle biopsy has no consequence on the mortality and body condition in fish as Russell's snapper Lutjanus russeli and Grass emperor Lethrinus laticaudis [START_REF] Henderson | Assessing the suitability of a nonlethal biopsy punch for sampling fish muscle tissue[END_REF]. However, growth rate of Sea bass declined after their muscle being biopsied [START_REF] Quéméneur | The relationships between growth rate and mitochondrial metabolism varies over time[END_REF]. Furthermore, muscle biopsy procedure might be expected to have important consequences for organismal traits that directly rely on bioenergetics, such as growth, locomotory activity, and whole-organism metabolism, but these consequences remain largely unexplored.

The aims of our study were (i) to measure the repeatability over time of mitochondrial traits as well as whole-animal performance parameters and (ii) to determine the consequences of muscle biopsy procedure on whole-animal performance of goldfish Carassius auratus. We conducted a longitudinal experiment to evaluate repeatability of red muscle mitochondrial traits 14 days apart (OXPHOS and LEAK respirations, as well as Basal respiration) using biopsied samples of red muscle, and whole organism performance measured three times 14 days apart (whole-organism metabolic rate and swimming performance, Figure 1). We evaluated the effect of the muscle sampling procedure on whole-animal performance with whole-organism metabolic rate (SMR and MMR) and swimming performance (optimal cost of transport -OptCOT and critical swimming speed -Ucrit). We also tested how intraspecific variation in organismal performance across the experimental period relates to variation in their mitochondrial metabolic properties. We analyzed mitochondrial properties in the red muscle, as it can be sampled using biopsy punches to collect a muscle tissue plug in a non-lethal manner [START_REF] Jeffries | The use of non-lethal sampling for transcriptomics to assess the physiological status of wild fishes[END_REF][START_REF] Porter | Resistance Exercise Training Alters Mitochondrial Function in Human Skeletal Muscle[END_REF] and has a major contribution in whole-animal oxygen consumption and aerobic swimming in fish [START_REF] Mckenzie | Swimming and other activities | Energetics of Fish Swimming[END_REF]. To date, muscle biopsy has only been employed in kilograms endotherm organisms (Humans [START_REF] Pesta | Chapter 3 -High-Resolution Respirometry: OXPHOS Protocols for Human Cells and Permeabilized Fibers from Small Biopsies of Human Muscle[END_REF]; King penguins [START_REF] Monternier | Mitochondrial phenotypic flexibility enhances energy savings during winter fast in king penguin chicks[END_REF]) with the sole exception of the recent study using a species of fish, the Sea Bass [START_REF] Quéméneur | The relationships between growth rate and mitochondrial metabolism varies over time[END_REF]. By doing so, we provide a detailed description of how to develop the biopsy procedure in organism for which the method has never been applied before, and we illustrate that the red muscle biopsy can be used in smaller animal as small as tens of grams, in our case ~30 g goldfish.

Material and Methods

Animals husbandry

In January 2022, goldfish juveniles (Carassius auratus, n = 32) were obtained from a commercial breeder (Anthias Aquariologie, Les Chères, France). Fish were transported to the University of Lyon 1 where they were held in two replicate 200L tanks (n = 16 experimental fish per tank) and allowed to acclimate for 11 days at room temperature (mean ± SD: 19.2 ± 0.3°C) in a continuously fully aerated water with a light cycle of 12h:12h. Fish were fed to satiety 6 days a week with commercial pellets (Tetra, Goldfish Granules). Fish were then randomly distributed in eight replicate 50L tanks (n = 4 per tank). To identify individual fish, variation in the body colour was used. Half of the water of each aquarium was replaced every week and water quality was monitored throughout the experiment with test strips (JBL Easy Test 6 in 1, JBL GMBH; ). Additional fish (n = 8 per tank) were present in the two initial tanks (final density: n = 24 per tank). These additional fish were used in pilot experiments (see the 'Preliminary studies' section below and results in the Supplemental data).

All experimental procedures were approved by the French ethical committee (APAFIS #34451-2021122117327959 v2).

Muscle biopsies

Fish were randomly assigned to two treatments: 'Biopsy' and 'Control'. The biopsiedfish experienced handling, anaesthesia and two biopsies, one on each flank 14 days apart. The non-biopsied (control) fish experienced handling and anaesthesia but no biopsy (Figure 1). The body mass, body length and Fulton index (an index of body condition calculated as 100 * (body mass / body length 3 ); [START_REF] Gilliers | Comparison of growth and condition indices of juvenile flatfish in different coastal nursery grounds[END_REF] at the start of the experiment were not different between the groups (Linear models: Body mass: P = 0.740; Body length: P = 0.680, Fulton index: P = 0.564, see Supplemental Data Section 1 Table S1).

Each fish was isolated in a 20L-aquarium and fasted for 24 hours before the procedure. On day of the procedure, anaesthesia and analgesia were performed on fish with a solution of MS-222 (150 mg/L) and lidocaine (5 mg/L) until loss of equilibrium and dyspnea (AVMA, 2020). The addition of an analgesic compound such as lidocaine was used to prevent animal's pain [START_REF] Sneddon | Clinical Anesthesia and Analgesia in Fish[END_REF].

Deeply anesthetized fish (total loss of equilibrium, slow and irregular opercular movement, see [START_REF] Hoseini | Anaesthetic efficacy of eugenol on iridescent shark, Pangasius hypophthalmus (Sauvage, 1878) in different size classes[END_REF] was immediately placed on a surgical table with a constant flow of the anaesthetic solution to the mouth throughout the surgical procedure (see Supplemental Data Section 2 Figure S1). A moistened tissue was placed over the body to prevent the animal dryness. Biopsy was performed on the lateral line to collect a sample of red muscle [START_REF] Rome | Why animals have different muscle fibre types[END_REF]. First, three scales were removed with forceps and the skin was opened over a 4mm-superficial line with a scalpel. The muscle was collected using a biopsy punch (Biopsy Punch, Kai medical, diameter: 1.5 mm) and the muscle sample (4.1 ± 0.5 mg) was immediately placed in a respiration buffer on ice (MiRO5: 0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, 1 g.L -1 free-fatty-acid bovine serum albumin, pH 7.1, Gnaiger, 2020). The wound was then sutured with a stitch (JV398, Vicryl) and a protective gelling powder (Orahesive, ConvaTec) was applied to the wound before the fish was placed in an oxygenated recovery bath (duration for full recovery from anaesthesia: 9 ± 5 min). When the fish was fully awake, it was placed back into the 20L aquarium in isolation for 4 hours before being placed back into its original aquarium. Control fish had no surgical procedure (i.e. no scales removed, no scalping and no biopsy) but remained on the surgical table with a continuous supply of anaesthetic solution to the mouth for a duration similar to those having the biopsy procedure (non-biopsied fish: 6 ± 1 min; biopsied-fish: 8 ± 2 min). Control fish were then placed in a recovery bath (8 ± 7 min) before being returned to their respective aquarium after 4 hours in isolation.

Whole-animal measurements: swimming performance and whole-animal metabolism

Each biopsied and control fish experienced three whole-animal performance measurements to determine their swimming performance (Ucrit and OptCOT) and whole-animal metabolism (SMR and MMR) 7-day before and 7-day after the biopsies (see Figure 1). The day before each Ucrit protocol, the fish were anaesthetized with MS-222 (100mg/L) and then were measured for body length and mass and placed in a 30L swim tunnel respirometer (Loligo Systems, Tjele, Denmark) over night at a speed of 0.5 body length per second (BL/s). The temperature was at the room temperature (17.9 ± 0.5°C). The fish were fasted for 24 hours before being placed in the respirometer. The oxygen consumption (ṀO2) of the fish was recorded from around 4pm to 8am the next day (minimum of 13 hours) and the standard metabolic rate (SMR) was calculated as the average 10% of the lowest oxygen consumption values [START_REF] Chabot | The determination of standard metabolic rate in fishes[END_REF].

The next morning, the Ucrit protocol was performed as in Thoral et al., 2022a. Individuals were exposed to a gradual increase in the current (0.5 BL/s every 30 minutes) until the fish was exhausted (resting on the bottom grid of the swimming area for more than 10 seconds; Thoral et al., 2022a). During the Ucrit protocol, the ṀO2 was also measured, and the maximum metabolic rate (MMR) was calculated as the maximum oxygen consumption. Ucrit (i.e. critical swimming speed) was calculated using Brett's equation [START_REF] Brett | The Respiratory Metabolism and Swimming Performance of Young Sockeye Salmon[END_REF]:

𝑈𝑐𝑟𝑖𝑡 = 𝑉𝑓 + * 𝑇𝑓 𝑇𝑖 , 𝑉𝑖
Where Vf is the last speed reached by the fish for which the fish swam during the complete speed step (BL/s), Tf is the time held at the last speed step reached (min), Ti is the full period of a speed step (30 min) and Vi is the speed increment between steps (0.5 BL/s).

The optimal cost of transport (OptCOT) in µg O2 cm -1 kg -1 of fish was calculated as the minimal cost of transport calculated during the Ucrit protocol:

𝐶𝑂𝑇 = / Ṁ𝑂2 𝑆𝑝𝑒𝑒𝑑 6 * 1000
With the ṀO2 expressed in mg O2 s -1 kg -1 of fish and the Speed in cm s -1 .

We verified whether control and biopsied fish did not differ in terms of whole-animal performance at the beginning of the experiment, on Day 0, using linear models (see Table S1).

At day 0, the SMR, Ucrit and OptCOT were not different between control and biopsied fish (LM: P > 0.05), while the MMR was higher in biopsied fish (LM: F = 5.018, P = 0.042).

Mitochondrial metabolism

Mitochondrial metabolism of biopsied red muscle was measured using high-resolution respirometers (Oxygraphs 2k high-resolution respirometers, Oroboros Instruments, Innsbruck, Austria). The respirometers were calibrated every morning at zero and air saturation oxygen levels. All measurements were done at 20°C and the data were acquired using DatLab v. 7.

After removing potential white muscle fibres under a binocular magnifying glass, the red muscle sample was gently dried on aluminium foil and then weighed before being placed in the respirometer chamber containing 2.1 mL of MiR05 without undergoing chemical or mechanical permeabilization. We have shown in previous studies that similar tissue preparation in fish allows for stimulation of respiratory fluxes by exogenous substrates [START_REF] Thoral | Changes in foraging mode SMR (mg O2 h -1 ) 3.05 ± 0[END_REF]Thoral et al., 2022a); we can thus assert that the plasma membranes in our experimental conditions were permeabilized.

A sequential substrate and inhibitor titration protocol, adapted from [START_REF] Teulier | Muscle bioenergetics of two emblematic Mediterranean fish species: Sardina pilchardus and Sparus aurata[END_REF]Thoral et al., 2022a, was immediately run for each fish. First, the Basal respiration was obtained following the addition of pyruvate (final concentration in the respirometry chamber:

5 mM), malate (2.5 mM) and succinate (5 mM). Then, the ATP synthase was activated with the injection of ADP (1 mM) to obtain the phosphorylating respiration (i.e., OXPHOS respiration).

Non-phosphorylating respiration was then measured (i.e., LEAK respiration) by adding oligomycin (1.25 µg/mL) that inhibited the ATP synthase. Finally, the residual oxygen consumption (i.e., ROX respiration) was determined by adding antimycin A (2.5 µM); ROX was then removed from all other respiration rates. One negative respiration value of Basal respiration has been subsequently removed from the analyses. The respiratory control ratio (RCR) has been calculated as the rate of respiration in presence of ADP and cytochrome c divided by the rate of respiration in presence of oligomycin. The mean RCR value was 3.22 (± 0.40), suggesting a good integrity of the inner mitochondrial membranes [START_REF] Chung | Intraspecific variation and plasticity in mitochondrial oxygen binding affinity as a response to environmental temperature[END_REF][START_REF] Fongy | Ontogeny of muscle bioenergetics in Adélie penguin chicks ( Pygoscelis adeliae )[END_REF][START_REF] Hedges | Voluntary wheel running prevents the acidosis-induced decrease in skeletal muscle mitochondrial reactive oxygen species emission[END_REF]. The integrity of the outer mitochondrial membrane was also tested by the injection of cytochrome c (0.01 mM) under OXPHOS state. The mean cytochrome c effect in the biopsied fish was 24% (± 3%).

The Net Phosphorylation Efficiency [START_REF] Shama | Transgenerational effects persist down the maternal line in marine sticklebacks: gene expression matches physiology in a warming ocean[END_REF] was calculated as follows:

𝑁𝑒𝑡 𝑃ℎ𝑜𝑠𝑝ℎ𝑜𝑟𝑦𝑙𝑎𝑡𝑖𝑜𝑛 𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 1 - 𝐿𝐸𝐴𝐾 𝑟𝑒𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛 𝑂𝑋𝑃𝐻𝑂𝑆 𝑟𝑒𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛

Preliminary studies

To evaluate the effect of lidocaine on mitochondrial metabolism, whole-animal metabolism and swimming performance, some fish were anaesthetized with lidocaine (n = 8) in addition to MS-222 while others were anaesthetized with no lidocaine (n = 8; See Supplemental Data section 3). Mitochondrial metabolism, whole-animal metabolism and swimming performance did not differ between fish exposed or not to lidocaine (Figure S3 and Figure S4).

In a separate group of goldfish (n = 8), a comparison in the mitochondrial metabolism between technics of tissue sampling was performed, and the technical repeatability of mitochondrial metabolic traits was assessed (see Supplemental Data section 4). These fish were anaesthetized and biopsied as above and, the day after, they were anaesthetized and euthanized (cervical dislocation) and dissected to collect red muscle. Collection of red muscle was performed by cutting a square of skin and removing a piece of red muscle with a scalpel as used in Thoral et al., 2022a. No effect of the sampling technique on the mitochondrial metabolic traits was found. However, the technical repeatability between the two red muscle samples from the same individual was low to moderate depending on the respiration rates (See Supplemental data section 4).

Statistical analyses

We examined the repeatability of mitochondrial parameters across a 2-week period (between the first and the second biopsy). To control for effects of body mass on the repeatability of mitochondrial parameters, we used the RptR package. RptR package [START_REF] Stoffel | rptR : repeatability estimation and variance decomposition by generalized linear mixed-effects models[END_REF] allows to examine whether repeatability between trials remained while accounting for individual body mass (Model: "Mitochondrial trait ~ Trial (Biopsy 1 or 2) * Body mass + (1|Individual)"). Repeatability was also assessed using Intraclass Correlation Coefficients (ICC, [START_REF] Koo | A Guideline of Selecting and Reporting Intraclass Correlation Coefficients for Reliability Research[END_REF].

We also examined the repeatability of whole-animal performance in biopsied fish between trial 1 and 2 to measure the effect of the first biopsy. RptR package was also used to examine whether repeatability between trials remained while accounting for variation in body mass for SMR and MMR expressed in mg O2.h -1 . Thus, the model was "Whole-animal metabolic rate (SMR or MMR) ~ Trial (1 or 2) * Body mass + (1|Individual)". The repeatability of SMR and MMR using ICC was performed on the body-mass normalized metabolic rates (expressed in mg O2.h -1 .kg -1 of fish). Then, the repeatability of whole-animal performance in biopsied fish has been assessed between trial 2 and 3 to measure the effect of the second biopsy as above. The repeatability of whole-animal performance over time has also been assessed in control (non-biopsied) fish.

Repeatability estimates with the RptR package are presented in the main document whereas ICC analyses are reported in the Supplemental Data Section 5 for the biopsied fish and Supplemental Data Section 6 for the control (non-biopsied) fish. We also performed paired ttests to determine whether mean values of mitochondrial and whole-animal parameters changed over the course of the experiment.

We also performed linear mixed models (LMM, Chi-square test [X²]) to test the effect of the biopsy procedure (fixed factor: biopsied or control fish) and time (fixed factor: day 7 and day 21) as well as their interaction, with individuals as a random effect, on the whole-animal traits (SMR, MMR, Ucrit, OptCOT, body mass and Fulton index). The body mass was also integrated as a covariate in the models including SMR and MMR as the dependant variable.

Thus, the model for the whole-animal metabolic rates was: "SMR or MMR ~ Treatment (control or biopsied) * Time (day 7 or 21) + Body mass + (1|Individual)" and the model for the other whole-animal traits was: "Trait ~ Treatment * Time + (1|Individual)". The model was then simplified according to a stepwise procedure by removing non-significant interaction and nonsignificant fixed effects (when not included in a significant interaction).

We finally tested correlation between mitochondrial and whole-animal parameters within the same fish by performing Pearson correlation tests. As all the results were not significant (P > 0.05), these results are presented in the Supplemental Data Section 7 (see Table S2).

The data from whole-animal measurements on day 28 for one biopsied fish were excluded from the experiment because the wound of this fish reopened during the swim. The whole-animal measurement data from one control fish on day 28 were missing because this fish died before day 28 (jump out of the aquarium). At the mitochondrial level, a negative value of Basal respiration due to a high ROX respiration was removed from the analyses in the biopsied fish anesthetized with MS-222 plus lidocaine. At the individual level, five SMR values are missing because of technical problems (n = 2 for control fish; n = 2 for biopsied fish anesthetized with MS-222 plus lidocaine; n = 1 for biopsied fish anesthetized with MS-222).

Each statistical analysis including mitochondrial parameters was run with and without individuals for which the cytochrome c effect was higher than mean ± 2SD. Statistical outcomes were not different when including or excluding high cytochrome c effect; all mitochondrial data were kept in the dataset. We ran supplemental analyses because of an extreme value of cytochrome c effect of 73% for a fish. The RCR value of the mitochondria from this fish was 2.44, showing a good integrity of the inner membranes. The patterns of the analyses of mitochondrial properties were the same whether or not this individual was included in the models, so models including mitochondrial data from this fish are reported in the manuscript.

A P-value was considered significant when ≤ 0.05. All statistical analyses were performed on R v. 4.0.3.

Results

I.

Repeatability of mitochondrial parameters between first and second biopsy.

Between the first and second biopsy, the repeatability was high for the Basal respiration (R = 0.959, P < 0.001; Figure 2A) and Net Phosphorylation Efficiency (R = 0.625, P = 0.024; Figure 2D), whereas the repeatability was moderate for the LEAK respiration (R = 0.406, P = 0.118;

Figure 2C) to low for the OXPHOS respiration (R = 0.061, P = 0.432; Figure 2B).

However, the mean mitochondrial parameters did not differ between biopsy 1 and 2 (Paired Ttest: all P > 0.05, Figures 2A to 2C), except for the Net Phosphorylation Efficiency that increased from the 1 st to the 2 nd biopsy (Paired T-test: t = -4.864, P = 0.002, Figure 2D).

II.

Repeatability of whole-animal performance in biopsied and control fish.

In the biopsied fish, the repeatability of SMR and MMR before and after the first biopsy was poor (Figure 3A and 3B, R = 0.126, P = 0.378 and R = 0, P = 1, respectively). However, the repeatability of Ucrit and OptCOT was high (Figure 3C and 3D, R = 0.643, P = 0.036 and R = 0.807, P = 0.006, respectively). We found no significant difference for mean values of SMR (Paired T-test: P = 0.469, t = 0.784), MMR (P = 0.633, t = -0.500), Ucrit (P = 0.098, t = 1.909)

and OptCOT (P = 0.427, t = -0.843) before and after the first biopsy.

In contrast, the repeatability of SMR before and after the second biopsy was moderate (Figure 3A, R = 0.201, P = 0.284), but the repeatability of MMR, Ucrit and OptCOT was null (Figure 3B to 3D, R = 0, P = 1). As above, we found no significant difference for mean values of SMR (P = 0.621, t = 0.517), MMR (P = 0.636, t = 0.498), Ucrit (P = 0.324, t = 1.074) and OptCOT (P = 0.546, t = -0.640) before and after the second biopsy.

In the control (non-biopsied) fish, the repeatability between the first and the second trial was low for the SMR (R = 0.09, P = 0.407, Figure S6A) and high for the MMR (R = 0.656, P = 0.023, Figure S6B) and OptCOT (R = 0.666, P = 0.015 Figure S6D), and the Ucrit tended to be repeatable, but not significantly (R= 0.65, P = 0.18, Figure S6C). Between the second and the third trial, the repeatability was null for the MMR (R = 0, P = 1; Figure S6B) and moderate (but not significant) for the SMR (R = 0.285, P = 0.239. Figure S6A). However, the repeatability was high for the OptCOT (R = 0.919, P < 0.001; Figure S6D) and (but not significant) for the Ucrit (R= 0.502, P = 0.077; Figure S6C). Moreover, we found no significant difference for mean values of SMR, MMR, Ucrit and OptCOT between the first and second trial, and also between the second and third trial (P > 0.05, Figure S6).

III.

Effect of biopsy procedure on whole-animal performance.

The body mass and Fulton index were not different between control and biopsied fish (LMM:

Body mass: X² = 0.056, P = 0.814; Fulton index: X² = 2.994, P = 0.084; Table 1). Moreover, we observed that the SMR, regardless of the trial, did not differ between the biopsied and control fish (X² = 0.289, P = 0.591). In addition, regardless of the biopsy procedure, fish did not differ in their SMR between day 14 and day 28 (X² = 0.211, P = 0.646). The MMR and the OptCOT were not affected by the biopsies (X² = 1.454, P = 0.228 and X² = 2.703, P = 0.100) and did not differ between day 14 and day 28 (X² =0.067, P = 0.796 and X² = 2.187, P = 0.139).

Finally, the Ucrit was also not affected by the biopsies (X² = 1.043, P =0.307) and by the measurement day (X² = 2.802, P = 0.094).

Discussion

In this study, we evaluated the repeatability of mitochondrial metabolism as well as whole-animal performance in goldfish, and we determined consequences of biopsy procedure for whole-animal performance. Interestingly, the mitochondrial Basal respiration and Net Phosphorylation Efficiency in red muscle were repeatable over time. However, even if repeatability of whole-animal metabolism and swimming performance seemed lowered by biopsy, our results show that two muscle biopsies made 14 days apart did not statistically affect the mean whole-animal performance parameters.

Several recent articles have raised important issues regarding the changes in mitochondrial traits over time [START_REF] Quéméneur | The relationships between growth rate and mitochondrial metabolism varies over time[END_REF][START_REF] Stier | Plastic but repeatable: Rapid adjustments of mitochondrial function and density during reproduction in a wild bird species[END_REF]. Our fish showed state-specific repeatability of the mitochondria, even with a stability of the environmental conditions in which the goldfish were living between measurements. Basal respiration rate was repeatable for a time interval of 14 days, whereas we found a lower repeatability in other traits such as LEAK and OXPHOS respirations. Basal respiration is limited by oxidation of energy substrates originally present in the cells, indicating that individual variation in the mitochondrial respiration from cellular substrates is stable over a 2-week time and within an environmental context. LEAK and OXPHOS respirations can rapidly change in mitochondrion resulting from new protein assembly in the respiratory chain [START_REF] Somero | Protein Assembly Regulation: Physiological and Perspectives Evolutionary[END_REF], or changes in phospholipid composition in the inner membranes [START_REF] Kraffe | Changes in mitochondrial oxidative capacities during thermal acclimation of rainbow trout Oncorhynchus mykiss: Roles of membrane proteins, phospholipids and their fatty acid compositions[END_REF]. In our study, it is possible that some individuals have been highly sensitive to the first biopsy, and they might have released high level of stress hormone such as corticoids that caused changes in the mitochondrial structure [START_REF] Duclos | Effects of corticosterone on muscle mitochondria identifying different sensitivity to glucocorticoids in Lewis and Fischer rats[END_REF]. There may also be a change in mitochondrial function associated with the fish handling with the measurement of whole-animal performance 7-day earlier, so that mitochondrial traits in some individuals are more flexible and can change in response to an intensive swimming test or isolation in a respirometry chamber. Although poorly studied, reaction norms of mitochondrial traits can differ among individuals in their direction as well as their magnitude. Thus, repeatability of mitochondrial traits may be low in variable environments because individuals might differ in their response to environmental change. In the present study, we overcome the analytical challenges of longitudinal sampling for mitochondrial assay, which offer future perspectives to explore the dynamics of mitochondrial function across conditions.

Our study showed that mitochondrial metabolism is measurable in red muscle biopsies performed in fish of a 10cm size and weighing less than 30g, with no consequence for their whole-animal performance. These results echoed those from [START_REF] Quéméneur | The relationships between growth rate and mitochondrial metabolism varies over time[END_REF] where one muscle biopsy did not affect the individual differences in the rank order of growth rate in Sea bass. The feasibility of measuring mitochondrial metabolism from tissue samples as small as milligrams of red muscle add on possibility in other types of tissues, such as liver [START_REF] Kuznetsov | Evaluation of mitochondrial respiratory function in small biopsies of liver[END_REF] and gills (Dawson et al., 2020). As samples can be collected without sacrificing animals, biopsy procedure might provide a non-terminal approach to determine the mitochondrial metabolism of wild animals. We shall first determine whether tissue biopsies affect animal performance on long term, and future studies will have to be done over a period longer than 14 days. Yet, our results are encouraging for future projects focusing on small animal species, such as great tits Parus major, which are of interest for projects looking at changes in mitochondrial metabolism in natural setting [START_REF] Casagrande | Increased glucocorticoid concentrations in early life cause mitochondrial inefficiency and short telomeres[END_REF][START_REF] Nord | Avian red blood cell mitochondria produce more heat in winter than in autumn[END_REF]. The biopsy of red muscle provide the means to move toward non-lethal sampling of animals, including small ectotherms, and toward ecological studies of mitochondrial metabolism [START_REF] Morgan | Are model organisms representative for climate change research? Testing thermal tolerance in wild and laboratory zebrafish populations[END_REF][START_REF] Morgan | Reduced physiological plasticity in a fish adapted to stable temperatures[END_REF]Touzot et al., 2019).

In our study, no significant relationship between the mitochondrial metabolism and whole-animal performance has been found. An explanation for this discrepancy might lie in the fact that rates of mitochondrial respiration are tissue-specific [START_REF] Farhat | Goldfish Response to Chronic Hypoxia: Mitochondrial Respiration, Fuel Preference and Energy Metabolism[END_REF][START_REF] Ost | Analysis of mitochondrial respiratory function in tissue biopsies and blood cells[END_REF] and the correlation of mitochondrial respiration rates across different tissues in the same individual can be poor [START_REF] Salin | Variation in metabolic rate among individuals is related to tissue-specific differences in mitochondrial leak respiration[END_REF][START_REF] Salin | Differences in mitochondrial efficiency explain individual variation in growth performance[END_REF]. The red muscle might thus not be representative of the overall rate of oxygen consumption and ATP production in the entire animal, because this would be defined as the sum of the individual tissue-specific rates. For instance, a recent study showed that mitochondrial metabolism in pectoral muscle was related to thermogenic capacity of capped chickadees whereas liver metabolism, but not the muscle one, was related to the bird BMR [START_REF] Milbergue | How does mitochondrial function relate to thermogenic capacity and basal metabolic rate in small birds ?[END_REF]. An alternative explanation is based on our sample sizes that might have been insufficient to detect statistically significant covariation in traits such as mitochondrial functioning, swimming performance and wholeanimal metabolic rates. It has previously been shown that a sample size of 28-40 fish when using a correlative approach allow to detect intraspecific variation in mitochondrial and whole animal performance [START_REF] Salin | Variation in metabolic rate among individuals is related to tissue-specific differences in mitochondrial leak respiration[END_REF][START_REF] Salin | Differences in mitochondrial efficiency explain individual variation in growth performance[END_REF][START_REF] Thoral | Changes in foraging mode SMR (mg O2 h -1 ) 3.05 ± 0[END_REF]. While measurement of variation among individual fish were a secondary scope of the present study, our work overcome an especially important hurdle for estimating individual variation of mitochondrial traits, and non-lethal measures in relatively large sample size will provide important insights in this area in coming years.

Conclusion

Our results showed that longitudinal and repeated sampling can be performed in a fish model as small as tens grams, testing for the first time how individual variation in mitochondrial metabolism is related to upcoming whole-organism performance. As shown in one of our previous studies (Thoral et al., 2022b), inter-individual variability in mitochondrial metabolism can be strongly affected by environmental conditions. Therefore, it seems essential, especially in a climate change context where individual responses will be more decisive than mean responses [START_REF] Bestion | Dispersal response to climate change: Scaling down to intraspecific variation[END_REF][START_REF] Fangue | Do mitochondrial properties explain intraspecific variation in thermal tolerance?[END_REF], to set up protocols that allow measuring metabolism of the same individuals at different times in order to assess its temporal evolution depending on environmental conditions. Our approach provides the means to move towards direct assessment of mitochondrial flexibility in individual animals from the size of tens grams.

Studying mitochondrial function with no need to cull animals is also a great advantage as it prevents animal sacrifice and improves statistical power [START_REF] Peters | Sample size requirements in trials using repeated measurements and the impact of trial design[END_REF]. Finally, analyses of mitochondrial function from biopsy sampling could be used to investigate the supposed relationship between mitochondrial and whole-animal metabolism across a range of ecological context, in the laboratory and ultimately in the wild, which are relevant criteria for ecologists and evolutionary biologists.

Figures

Figure 1: Experimental protocol description. Whole-animal performance was estimated from measurements of whole-animal oxygen consumption rate (ṀO2) and swimming speed (Ucrit) and repeatability was evaluated three times across a 28-day period at 14-days interval. Biopsied fish had two biopsies of red muscle for measurement of mitochondrial metabolism.

Repeatability of the biopsy was evaluated two times across 14-days interval, which occurred between whole-animal performance measurements. The control (non-biopsied) fish experienced handling and anaesthesia procedure, as well as whole-animal performance measurements similar to biopsied-fish, but no biopsy.

Figure 2 .

 2 Figure 2. Repeated measurements of mitochondrial parameters at 14-day interval. The mean ± sem is shown (dark dots) as well as the individuals measured in the two biopsies (light dots).**Indicates a significant difference between biopsies 1 and 2 (**P < 0.01).

Figure 3 :

 3 Figure 3: Repeated measurements of whole-animal metabolism and swimming performance in biopsied fish at trial 1, 2 and 3. Dashed lines indicated the biopsies. SMR (A): Standard metabolic rate; MMR (B): Maximum metabolic rate, Ucrit (C): critical swimming speed; OptCOT (D): optimal cost of transport. The mean ± sem is shown (dark dots) as well as the individuals measured in the two whole-animal performances (light dots).

Acknowledgements

We are grateful to Anthias Aquariologie for providing goldfish. We would like to thank Lilian Redon for his help during the experiments, and Jean-Baptiste Quéméneur for constructive discussion about biopsy methods.

Funding

I.M-S. was supported by an ERASMUS mobility fellowship. Swim tunnels were partly supported by ZABR (Zone Atelier Bassin du Rhône) and Contrat de Plan Etat -Région Auvergne Rhône Alpes 2015-2021 through the project SedAqua.

Authors' contribution

LT and KS conceptualized the project and designed the methodology. LD and IM-S collected the data with the help of LT, ET, AM, AC, LA and JS. ET analysed the data. ET and LT led the writing of the manuscript and all authors contributed to the drafts and final approval for publication.

caused by a decline in prey size have major bioenergetic consequences for a small pelagic fish. J. Anim. Ecol. 90, 2289-2301. Weibel, E. R. and Hoppeler, H. (2005). Exercise-induced maximal metabolic rate scales with muscle aerobic capacity. J. Exp. Biol. 208, 1635-1644. Tables Table 1. Mean changes in whole-animal traits in biopsied and control (non-biopsied) fish for trial 2 and trial 3. SMR: Standard metabolic rate. MMR: Maximum metabolic rate. Ucrit: critical swimming speed. OptCOT: optimal cost of transport.