Pernille Jason A Greenbaum

Haste Andersen

Martin Blythe

Huynh-Hoa Bui

Raul E Cachau

James Crowe

Matthew Davies

A S Kolaskar

Ole Lund

Sherrie Morrison

Pernille Haste Andersen

Brendan Mumey

Yanay Ofran

Jean-Luc Pellequer

Clemencia Pinilla

Julia V Ponomarenko

G P S Raghava

Marc H V Van Regenmortel

Erwin L Roggen

Alessandro Sette

Avner Schlessinger

Johannes Sollner

Martin Zand

Bjoern Peters

Towards a consensus on datasets and evaluation metrics

Keywords: B-cell epitopes, epitope prediction, algorithms, immunology databases, data standardization, bioinformatics, software tools, tool development

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INTRODUCTION

Antibodies are proteins produced by B-cells in response to immunogenic substances such as viruses, allergens, and vaccines. B-cell, or antibody, epitopes are the molecular structures within an antigen that make specific contacts with residues of soluble and membrane-bound antibody molecules (See Figure 1 for a schematic introduction to key terms). For various immunological applications, a computational prediction of epitopes in an antigen is highly desirable. Many such tools exist that mainly base their predictions on shared amino acid characteristics of known B-cell epitopes in protein antigens. However, in contrast to predictions used to identify T-cell epitopes [START_REF] Brusic | Computational methods for prediction of T-cell epitopes-a framework for modelling, testing, and applications[END_REF][START_REF] Groot | Immunomics: discovering new targets for vaccines and therapeutics[END_REF], the quality of these predictions is widely considered to be too poor to be employed as a reliable tool by immunologists [START_REF] Blythe | Benchmarking B cell epitope prediction: underperformance of existing methods[END_REF]. From this point forward, the term 'epitope' shall refer to a B-cell epitope, unless otherwise specified.

In a workshop sponsored by the National Institute for Allergy and Infectious Diseases (NIAID) on 7 and 8 September 2006, a panel of immunologists and bioinformaticians convened in Washington, DC to discuss the current state and future directions for the epitope prediction field. The specific goals of the workshop were to review currently available epitope prediction tools, agree upon metrics to evaluate tool performance, identify a body of relevant training and test data, and develop recommendations for advertising and implementing these suggestions for the broader research community. This meeting report is meant to publicize a summary of the presentations and discussions at the workshop, and elicit community feedback, via a web-based forum (http://www.immuneepitope.org/ jive/), on the specific proposals and recommendations.

B-cell epitope terminology

Dr Marc H. V. van Regenmortel opened the workshop with an overview of epitope terminology issues. It was noted that researchers classify epitopes as either continuous or discontinuous (Figure 2), but many so-called discontinuous epitopes consist of stretches of several consecutive amino acids that could, in some cases, be considered continuous epitopes in their own right [START_REF] Van Regenmortel | Immunoinformatics may lead to a reappraisal of the nature of B cell epitopes and of the feasibility of synthetic peptide vaccines[END_REF]. Further, the distinction between linear and conformational epitopes is problematic, since linear peptides necessarily adopt a particular three-dimensional conformation that is recognized by their cognate antibody. Dr van Regenmortel also emphasized the need to clarify the purpose of making a specific epitope prediction, and how this clarification could direct selection of the most appropriate prediction tool or development of a new tool, as needed. For instance, if the purpose is to predict epitopes as vaccine candidates, then the predicted epitopes should elicit antibodies that recognize the vaccine target and could possibly provide protection from infection [START_REF] Van Regenmortel | Antigenicity and immunogenicity of synthetic peptides[END_REF]. However, if the purpose is to predict epitopes that could be used to replace complete infectious antigens in diagnostic immunoassays, the predicted epitopes should be able to react with antibodies found in hosts infected with the pathogenic agent. Since any given purpose may apply only to a subset of epitopes, the main goal of the prediction should be taken into account in selecting an appropriate dataset for tool training and evaluation.

The influence of antibody structure on its function was described in the context of responses to rotavirus infections by Dr James Crowe [START_REF] Crowe | Genetic and structural determinants of virus neutralizing antibodies[END_REF]. Central to the notion of an epitope is the understanding that epitopes are context dependent. That is, they are dependent entities and therefore cannot exist without a corresponding antibody. At the same time, genes encoding antibodies undergo a rapid process of somatic hypermutation during germinal center reactions resulting in variations of their binding site. It may therefore be useful to think of a protein surface as a continuous landscape of epitopic regions without welldefined borders. Under a given set of circumstances, any region of this landscape can behave as an epitope. By this definition, a very large ensemble of epitopes exists on any given protein surface. To complicate matters further, a portion of a protein surface that acts as an epitope under one set of circumstances (e.g., assay type, antibody concentration, antibody species specificity) will not necessarily behave as an epitope under another set. Therefore, binary classification of antigen regions into epitopes or non-epitopes may not accurately reflect biological reality. In an immunization (left panel), the host species is exposed to a substance that elicits the production of antibodies by B-cells. This substance is referred to as the immunogen, while the substance recognized by antibodies is referred to as the antigen. While antigen and immunogen are typically the same substance during the course of an infection, they are often distinct in an epitope mapping experiment. Such an experiment is depicted in the right panel, where different fragments or mutants of the immunogen are used as antigens in order to identify the specific antibody binding sites, which are referred to as epitopes.

B-cell epitope datasets currently available and under development

Defining the requirements for assembling relevant datasets was a recurring theme at the workshop. Martin Blythe described the data collected in the AntiJen database [START_REF] Toseland | AntiJen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data[END_REF] (Table 1), which consists of 3541 datapoints and is one of the largest available resources of linear and conformational epitope information. A dataset consisting of 50 linear epitope-mapped protein sequences was derived from the database [START_REF] Blythe | Benchmarking B cell epitope prediction: underperformance of existing methods[END_REF]. Several other groups have utilized these information resources for tool development and testing purposes. Other public data sources that were used for tool development and evaluation include: the comparably small, but high-quality, dataset of Dr Jean-Luc Pellequer [START_REF] Pellequer | Correlation between the location of antigenic sites and the prediction of turns in proteins[END_REF], consisting of 82 well-defined continuous epitopes across 14 proteins that have been studied extensively; the HIV Molecular Immunology Database, hosted at Los Alamos National Labs [START_REF] Korber | HIV Molecular Immunology[END_REF]; and the Bcipep database by Dr G. P. S. Raghava's group [START_REF] Saha | Bcipep: a database of B-cell epitopes[END_REF], containing 3031 epitopes.

The approach towards assembling epitope datasets from Immune Epitope Database (IEDB) [START_REF] Peters | The immune epitope database and analysis resource: from vision to blueprint[END_REF] was presented by Dr Alessandro Sette. The IEDB contains highly annotated T-and B-cell epitopes, from which users can select datasets relevant to their particular purpose. For example, epitopes identified by immunization with the native antigen and assayed with peptides can be considered separately from those immunized with peptides and assayed with native antigen. Along with epitopes identified using functional assays, the IEDB includes curated data on epitopes inferred from three-dimensional structures of antigen-antibody complexes available in the Protein Data Bank (PDB) [START_REF] Berman | The Protein Data Bank[END_REF]. The database also includes a large volume of negative data; that is, peptide and protein sequences experimentally shown not to be recognized by antibodies in a particular assay. It should be emphasized that these regions may, in fact, behave as epitopes when sampled under a different set of conditions, or with a different population of antibodies. To provide user-friendly datasets for epitope prediction tool development and testing, the IEDB team is in the process of assembling all relevant, non-redundant data into several customized datasets, which will be available to the public by mid-2007.

B-cell epitope prediction tools

Currently available tools and their evaluations were discussed by Dr Jean-Luc Pellequer. Specifically, the [START_REF] Underwood | An antigenic map of the haemagglutinin of the influenza Hong Kong subtype (H3N2), constructed using mouse monoclonal antibodies[END_REF] represented as ribbons. In this case, the epitope consists of residues that are distant in the primary sequence, but close when the protein is folded into its native three-dimensional structure. All of the residues are required for recognition by the antibody and thus are not epitopes on their own. (d) Surface rendering of epitope shown in (c). This rendering elucidates how residues that are distant in the primary sequence can be part of the same epitope.

distinction between a database and a dataset was elucidated. The former serves as the data warehouse and contains all potentially relevant data, while the latter is a smaller subset of data intended for tool development purposes. Following this general introduction, various currently available tools for epitope prediction were presented and are briefly summarized and loosely categorized here: sequence-based tools-tools that take only amino acid sequence information as input; structure-based tools-tools that require some form of three-dimensional structure information; and tools incorporating data from phage-display experiments.

SEQUENCE-BASED PREDICTION TOOLS

The 'classical' approach to epitope prediction is to utilize amino acid propensity scales such as hydrophilicity or chain flexibility to identify regions in antigens that are likely to contain epitopes. One such approach is performed by the BEPITOPE tool [START_REF] Odorico | BEPITOPE: predicting the location of continuous epitopes and patterns in proteins[END_REF], presented by Dr Pellequer, which makes use of the predictive power of a consensus voting method based on the prediction of turns in proteins. A consensus method that combines several different prediction methods is also available in BEPITOPE. Additionally, several scale-based epitope prediction tools have been made available on the IEDB website. Evaluation of these tools, using datasets from Pellequer [START_REF] Pellequer | Correlation between the location of antigenic sites and the prediction of turns in proteins[END_REF] and the AntiJen [START_REF] Toseland | AntiJen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data[END_REF] and HIV databases, gave area under the receiver operating characteristic curve (A ROC ) [START_REF] Swets | Measuring the accuracy of diagnostic systems[END_REF] values around 0.60, similar to those reported by other groups for amino acid scales. Briefly, A ROC scores range from 0 to 1, with a score of 0.5 equal to random discrimination, and 1 equal to perfect performance [START_REF] Lund | Immunological Bioinformatics[END_REF]. The most detailed evaluation of such scales was presented by Martin Blythe [START_REF] Blythe | Benchmarking B cell epitope prediction: underperformance of existing methods[END_REF], who tested 484 amino acid scales from the AAindex database [START_REF] Kawashima | AAindex: Amino Acid Index Database[END_REF]. The combination of scales and experimentation with several machine-learning algorithms showed little improvement over single scale-based methods, which were considered to perform inadequately. Machine-learning tools attempt to extract characteristics of an epitope from a set of learning examples, and generalize them in a classification algorithm. One such approach using an artificial neural network (ANN) was applied by [START_REF] Saha | Prediction of continuous B-cell epitopes in an antigen using recurrent neural network[END_REF] in the ANN-Based B-cell Epitope Prediction (ABCpred) algorithm. The system was trained on 700 epitopes from the Bcipep database and 700 randomly selected peptides represented by amino acid sequences of lengths varying between 10 and 20 amino acids. Employing fivefold cross-validation on this dataset, the method achieved a maximum accuracy of $66%. Bepipred [START_REF] Larsen | Improved method for predicting linear B-cell epitopes[END_REF], an algorithm that combines scores from the Parker hydrophilicity scale [START_REF] Parker | New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and X-ray-derived accessible sites[END_REF] and a hidden Markov model (HMM) trained on linear epitopes, was presented by Dr Ole Lund. The method shows a small, but significant, increase in A ROC over earlier scale-based methods. The sequence parametrizer (SPA) algorithm [START_REF] Sollner | Selection and combination of machine learning classifiers for prediction of linear B-cell epitopes on proteins[END_REF][START_REF] Sollner | Machine learning approaches for prediction of linear B-cell epitopes on proteins[END_REF], along with its associated machine-learning methods, was presented by Dr Johannes Sollner. In addition to using the common single amino acid propensity scales, this method also incorporates neighborhood parameters reflecting the probability that a given stretch of amino acids exists within a predefined proximity of a specific amino acid residue. Training and testing on epitope sequences pulled from a high-quality proprietary database, as well as several publicly accessible databases, yields a degree of accuracy that is greatly increased over single-parameter methods.

STRUCTURE-BASED PREDICTION TOOLS

Several groups presented novel tool development efforts taking advantage of three-dimensional structures of antigen-antibody complexes available in the PDB. The DiscoTope algorithm (Haste [START_REF] Andersen | Prediction of residues in discontinuous B-cell epitopes using protein 3D structures[END_REF], presented by Pernille Haste Andersen, is a sequence-structure hybrid method. A log-odds probability matrix of each amino acid residue taking part in an antigenic interaction was calculated based on data compiled from antigen-antibody complexes. This innovative algorithm combines scores from this matrix with a measure of the surface area to predict the location of discontinuous epitope residues and does so with a fair degree of accuracy. Using the A ROC as an indicator, the algorithm achieves a score of 0.711.

Another method employing the three-dimensional structure of the antigen for prediction of epitopic regions was presented by Dr A. S. Kolaskar. The conformational epitope prediction (CEP) [START_REF] Kulkarni-Kale | CEP: a conformational epitope prediction server[END_REF] server calculates the relative accessible surface area (RSA) for each residue in the structure and determines which regions of the protein molecule are sufficiently exposed to act as antigenic determinants. Additionally, regions distant in the primary sequence, but close in three-dimensional space are condensed into one epitope. To test the tool, in a dataset consisting of 63 antigen-antibody complexes, the algorithm correctly identified 76% of the epitopic residues.

Antigen-antibody complexes from the PDB were annotated and compiled into a database (Epitome) [START_REF] Schlessinger | Epitome: database of structure-inferred antigenic epitopes[END_REF] by Drs Yanay Ofran and Avner Schlessinger. Using structural alignments of antibodies bound to different antigens, the antibody residues that are part of the complementarity-determining region (CDR) could be defined. Contacts to this region are thought to play a role in the specific recognition of an epitope by an antibody. Consolidation of data in this manner allowed the authors to observe several interesting features at the sequence level of antigens. Specifically, compared to standard protein-protein interactions, the antigenic residues involved in antigen-antibody interactions exhibit a lower degree of conservation than residues involved in other protein-protein interactions. This finding, coupled with several other sequence factors, could help to predict antigenic sites through sequence analysis.

Non-redundant datasets of representative threedimensional structures of protein antigens and antigen-antibody complexes from the PDB were assembled by Dr Julia Ponomarenko. These datasets were used to determine the sensitivity of antibody binding site prediction of several tools developed for various purposes. Established tools for epitope prediction (CEP), protein-protein interaction prediction [PPI-PRED [START_REF] Bradford | Improved prediction of protein-protein binding sites using a support vector machines approach[END_REF], ProMate [START_REF] Neuvirth | ProMate: a structure based prediction program to identify the location of protein-protein binding sites[END_REF]], and protein-protein docking [ClusPro [START_REF] Comeau | ClusPro: an automated docking and discrimination method for the prediction of protein complexes[END_REF], PatchDock [START_REF] Duhovny | Proceedings of the 2nd Workshop on Algorithms in Bioinformatics[END_REF]] were included in the study. While in this comparison, varying evaluation metrics showed that none of the tested methods demonstrated a very high degree of accuracy, the protein-protein docking algorithms yielded the highest sensitivity (56%). This underperformance can most likely be attributed to the fact that most of these methods were not designed for the purpose of epitope predictions. Consistent with the findings of the DiscoTope and Epitome studies, the comparison of sequence characteristics within epitopes to other surface residues, revealed a significantly lower degree of conservation, as well as significant differences in the frequency of individual residues and secondary structural elements.

TOOLS INCORPORATING PHAGE-DISPLAY DATA

One important and pervasive experimental technique to map epitopes is the use of phage-display libraries [START_REF] Smith | Phage display[END_REF]. By selecting phages from a library for their ability to bind antibodies specific for a known antigen, linear peptide sequences that cross-react with these antibodies, commonly referred to as mimotopes [START_REF] Meloen | Mimotopes: realization of an unlikely concept[END_REF], can be discovered. The exact linear sequence identified will generally not exist as a linear sequence in the antigenic protein. The identification of the region of the antigen mimicked by one of these peptides therefore requires special tools that, in turn, provide insight into antigen-antibody binding interactions.

Dr Erwin L. Roggen presented work on his Epitope Mapping Tool (EMT) [START_REF] Batori | An in silico method using an epitope motif database for predicting the location of antigenic determinants on proteins in a structural context[END_REF] that scrutinizes an antigen for the presence or absence of amino acid motifs commonly found in epitopes. Epitopic motifs were compiled by the alignment of sequences captured by the competitive immunoscreening of phage-display libraries. Combining the detection of these epitopic motifs with evaluations of the relative surface accessibility of residues in an antigen yielded predictions of the antigenicity of residues in food (e.g., lysozyme, Ara h 1, Ara h 2, Ara h 3), environmental (Der p 1, Bet v 1), and technical (industrial proteases) allergens that were in close agreement with experimental data.

The EPIMAP [START_REF] Mumey | A new method for mapping discontinuous antibody epitopes to reveal structural features of proteins[END_REF][START_REF] Mumey | Proceedings of High Performance Computing in Genomics[END_REF] method was presented by Dr Brendan Mumey. In this method, mimotopes discovered by phage display are individually aligned against their parent antigen via a dynamic programming algorithm. Resultant plots of the frequency of alignment for each residue of the parent protein can reveal segments that are close in three-dimensional space in the native fold. Application of this technique to IL-10 mimetic peptides has yielded residue classifications consistent with an experimentally mapped epitope.

Peptide sequences collected from the biopanning of phage libraries were mapped to three-dimensional structures by Dr Raul E. Cachau [START_REF] Cachau | A new addition to the structural bioinformatics toolbox: 3D models of short bioactive peptides from multiple sequences using feedback restrained molecular dynamics (FRMD)[END_REF]. This was accomplished by applying feedback restrained molecular dynamics (FRMD) to the peptide pool, under the assumption that all the mimotopes should be able to adopt a similar conformation. Validation was performed on an erythropoietin analog and the model was shown to be in good agreement with available structural data.

WORKSHOP RECOMMENDATIONS

Following formal presentations, workshop participants were asked to provide recommendations on the following topics:

Assembly of ideal datasets, including detailed information regarding dataset characteristics. Methods and metrics for tool evaluation. Resource needs and action items.

Characteristics of an ideal dataset

On a number of discussed issues, a broad but not necessarily unanimous consensus could be reached. One such issue was the assembly of a high-quality dataset for the training and testing of current and novel prediction algorithms. The quality of the underlying dataset is intrinsically linked to the predictive ability of the tools. In turn, the accessibility to a database of well-curated epitope data is essential to the development of a reliable dataset. This distinction between the database, which includes all possible information, and the dataset, which is a subset of relevant data from the database, is subtle, but critical to comprehend. Ideally, a database should include the following information: sequence information characterizing the epitope and antigen, the relationship between recognition of epitope and antigen (i.e., does immunization with the epitope produce antibodies capable of recognizing the native antigen and, conversely, does immunization with the antigen produce antibodies that can recognize epitopes contained within the antigen); the conformation in which the antigen is tested (i.e., native vs. non-native); the location of the epitope within the three-dimensional structure of the source antigen (if available); the host species producing the antibody, a classification of the antibody as functional (i.e., neutralizing or protective); and quantitative data on the interaction between epitope and antibody (e.g., binding affinity, frequency of recognition, etc.). Importantly, a database should also capture negative data, (i.e., regions of antigens that were experimentally tested and shown not to be recognized by antibodies). It should be stressed that incorporation of negative data in the training and testing datasets is as vital as the inclusion of epitope data, and should therefore be adequately represented. This type of database allows for the assembly of several training and testing datasets, one for use with each particular type of application. As alluded to earlier, the purpose of any specific prediction algorithm will dictate the proper dataset upon which it should be trained and evaluated.

As the datasets naturally expand, the establishment of a central public repository is desirable for direct comparison of tool performance, and to avoid unnecessary duplication of work. The IEDB can provide non-exclusive online hosting for such datasets, allowing all tool developers and immunologists to deposit their datasets onto a central server. At the same time, all content from the IEDB is free for download and redistribution in any form. Several databases and datasets, with unique and useful characteristics, were created before the IEDB, and likely will continue to be created and maintained. The IEDB developers propose that the IEDB can act as a central repository to facilitate the exchange of information by providing access to these datasets and proper credit to their creators. This arrangement will allow for several publicly accessible datasets to evolve in parallel, eventually leading to a community consensus on the best and most appropriate datasets for tool training and evaluation.

Metrics for tool evaluation

The purpose of tool evaluations is twofold: (1) Tool developers need a statistically sound assessment of tool prediction quality that allows for the comparison of current tools and aids in the development of new ones. The broad array of metrics currently used to report tool performance illustrates the need for a common measure. It was generally agreed that the most appropriate metric for tool performance evaluation is the A ROC . This measure has the advantage of being non-parametric and does not require a threshold for determining a positive prediction. The range of values is easily interpreted, with a score of 0.5 equal to random performance and 1 corresponding to perfect performance.

(2) Tool users need an easily understandable assessment of the level of accuracy that can be expected when making a prediction using a giving tool. This need makes additional metrics necessary, such as sensitivity, specificity, and positive predictive value. In addition to providing metrics that assess tool performance based upon a standard dataset, a narrative must be presented to tool users that explains their meaning and implications for biological applications.

The automated evaluation of tools on a regular basis using a standard dataset and set of metrics was discussed. This approach was generally thought to be a useful pursuit, as it has been an asset to the protein secondary structure prediction [START_REF] Koh | EVA: evaluation of protein structure prediction servers[END_REF] and transmembrane helix prediction communities [START_REF] Kernytsky | Static benchmarking of membrane helix predictions[END_REF]. To employ an automated server of this type, it is imperative that each tool incorporates the ability to import and export data in a common format. This standardization would not only facilitate the development of an evaluation server, but also the development of a metaserver for the user community that would act as a front end to the tool set. The ability to run related tools from one central server has proved invaluable in other fields [e.g., protein structure prediction [START_REF] Bujnicki | Structure prediction meta server[END_REF] and protein domain prediction [START_REF] Saini | Meta-DP: domain prediction meta-server[END_REF]], and would greatly increase user productivity. Citation issues have previously been a hindrance to development of this type of server, but the unquestionable value of such a resource ensures a resolution. It was agreed that each tool contributor should be included as an author in the publication describing such a server.

Data formats

As mentioned previously, standardization of data formats is desirable for the epitope datasets as well as for the input and output of the diverse tools. We propose to develop a format suitable for the representation of B-cell epitope information which is flexible enough to be easily interpreted by computers and convertible into existing or user-specified formats. This would allow users to import B-cell epitope data into pre-existing programs for data analysis, as opposed to building applications de novo. For example, converting to a genome browser compatible format, such as General Feature Format (GFF) would allow for the visual comparison of multiple algorithms on a mature platform as well as the calculation of metrics commonly used in genome annotation such as enrichment values. This type of interoperability can easily be achieved with, for example, a well-developed XML schema.

Another key advantage of this format is that it is capable of capturing both sequence and structure information, including interacting residues. Several different structural definitions of what constitutes the antibody binding site in an antigen are used by different groups. Differing opinions exist regarding which residues in an antibody are relevant and what intermolecular distance is used to define an interaction. The solution for the time being is to use multiple definitions; however, these are issues that must be resolved in the future.

CONCLUSIONS

It is clear from the presentations and discussions at this workshop that the current state of B-cell epitope prediction is far from ideal and that the ultimate need of the user community is access to better prediction tools. However, in order to develop more accurate tools, it is imperative that the recommendations for assembling high-quality training and testing datasets be followed. Additionally, the periodic re-evaluation of each tool's performance on these datasets according to a common metric is vital. This approach will allow tool developers to draw upon the most effective aspects of each algorithm in order to evolve a superior one. The implementation of the recommendations developed at this workshop will greatly enhance the development of epitope prediction tools. We would hope that the grounded approach outlined here would garner the confidence of the immunology community and would provoke experimental researchers to employ these tools in their own work. Ultimately, this report should be considered a call for action and feedback from the greater community on the proposals drafted here. As mentioned previously, we will provide a web-based forum for this purpose (http://www.immuneepitope.org/jive/) and we explicitly welcome any contributions or collaborations as a result.

Figure 1 .

 1 Figure 1. B-cell epitope mapping schematic. The figure schematically depicts an epitope mapping experiment.In an immunization (left panel), the host species is exposed to a substance that elicits the production of antibodies by B-cells. This substance is referred to as the immunogen, while the substance recognized by antibodies is referred to as the antigen. While antigen and immunogen are typically the same substance during the course of an infection, they are often distinct in an epitope mapping experiment. Such an experiment is depicted in the right panel, where different fragments or mutants of the immunogen are used as antigens in order to identify the specific antibody binding sites, which are referred to as epitopes.

Figure 2 .

 2 Figure 2. Continuous versus discontinuous epitopes. This figure contrasts the difference between a continuous and discontinuous epitope in Hemaglutinin precursor protein (Swiss-Prot ID: P03435, PDB code: 1HA0) of Influenza A virus. In each rendering, the residues that are part of the epitope are colored in cyan, while the remaining residues are gray. (a) Continuous epitope (Churchill et al., 1994) represented as ribbons. Here, the epitope sequence lies in one region of the protein. This is the minimal sequence required for recognition by the antibody. (b) Surface rendering of epitope shown in (a). (c) Discontinuous epitope[START_REF] Underwood | An antigenic map of the haemagglutinin of the influenza Hong Kong subtype (H3N2), constructed using mouse monoclonal antibodies[END_REF] represented as ribbons. In this case, the epitope consists of residues that are distant in the primary sequence, but close when the protein is folded into its native three-dimensional structure. All of the residues are required for recognition by the antibody and thus are not epitopes on their own. (d) Surface rendering of epitope shown in (c). This rendering elucidates how residues that are distant in the primary sequence can be part of the same epitope.

Table 1 .

 1 Tools, databases, and datasets

	Name	URL/email	Description
	ABCpred	http://www.imtech.res.in/raghava/abcpred	Sequence-based machine-learning tool for the
			prediction of continuous epitopes
	AntiJen Ã	http://www.jenner.ac.uk/AntiJen/	Database of binding data for various types of
			proteins, including B-cell epitopes and
			antibodies
	Bcipep Ã	http://www.imtech.res.in/raghava/bcipep/	Database of B-cell epitopes of varying
			immunogenicity
	Bepipred	http://www.cbs.dtu.dk/services/BepiPred	Sequence-based tool for the prediction of
			continuous epitopes
	BEPITOPE	jlpellequer@cea.fr	Sequence-based tool for the prediction of
			continuous epitopes
	CEP	http://bioinfo.ernet.in/cep.htm	Structure-based tool for the prediction of
			continuous and discontinuous epitopes
	DiscoTope	http://www.cbs.dtu.dk/services/DiscoTope	Sequence/structure-based tool for the
			prediction of discontinuous epitopes
	EMT	elro@novozymes.com	Phage-display based tool for the prediction of
			continuous and discontinuous epitopes
	EPIMAP	mumey@cs.montana.edu	Phage-display based tool for the identification
			of discontinuous epitopes
	Epitome	http://www.rostlab.org/services/epitome	Database of antigenic residues and interacting
			antibodies including detailed descriptions and
			visualization capabilities
	HIV database Ã	http://hiv-web.lanl.gov/content/immunology/	Database of HIV-specific immune epitopes
	IEDB ÃÃ	http://www.immuneepitope.org	Database of T-and B-cell epitopes and
			non-epitopes
	IEDB B-cell epitope tools http://www.immuneepitope.org/tools/bcell/iedb_input Sequence-based tool for the prediction of
			continuous epitopes
	Pellequer dataset	jlpellequer@cea.fr	Dataset consisting of 82 epitopes in 14 protein
			sequences
	SPA	johannes.soellner@emergentec.com	Program for the parametrization of peptide
			sequences. Intended for use with
			machine-learning algorithms

Copyright # 2007 John Wiley & Sons, Ltd. J. Mol. Recognit. 2007; 20: 75-82 DOI: 10.1002/jmr

Acknowledgments

The authors would like to acknowledge the NIAID, in particular Dr Alison Deckhut Augustine for critical review of the manuscript and the Division of Allergy, Immunology and Transplantation for providing organizational and funding support for the workshop participants. Molecular graphics images were produced using the UCSF Chimera package (Pettersen et al., 2004) from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). Kirin publication number 818.

, and prediction tools. Where URLs are not available, author's email addresses are supplied. Ã A dataset for tool training and testing has been assembled from this database and is available at the listed URL.


K Pasquon

S J Conway
email: susan.conway@univ-nantes.fr

M Vincendon

M Massé

J Raack

A Noblet

A Grau Galofre

Morino

G Munaretto

A Lucchetti

M Pajola

Stephen R Lewis

The Cassis

Insights into

Introduction 48

1.1. Gullies and seasonal frost et al., 2001), to sub-centimetre in thickness at lower latitudes [START_REF] Raack | Present-day 867 seasonal gully activity in a south polar pit (Sisyphi Cavi) on Mars[END_REF]. Most ice deposits are 85 composed of CO2 (~centimetres) (Diniega et al., 2013;Dundas et al., 2015), whereas H2O is a minor material within gully channels via small dry flows on top of the ice. When a critical mass sediment is 113 reached on top of the slab ice, the sediment flows down the still frosted gully on top of the sublimating 114 ice or as a mixture of dry material and sublimating ice as a flow. This accumulation phase means more 115 sediment would be mobilised, explaining the observable thickness of the flow deposits. Previous studies [START_REF] Raack | Present-day 867 seasonal gully activity in a south polar pit (Sisyphi Cavi) on Mars[END_REF]2020) showed that the activity of gullies in Sisyphi Cavi occurs 154 before the seasonal frost disappears from the surface, and thus suggest that a relationship likely exists 155 between gully activity and springtime defrosting. Here we perform a detailed study of seasonal frost 156 evolution on the slopes of Sisyphi Cavi (68-74°S; 345°-5°E) (Fig. 1A), exploring the relationship 157 between gully activity and frost occurrence. We focus on two main points: 1) Investigating the changing 158 appearance of the seasonal frost through the winter and spring before gully activity occurs, to build 159 intuition on the environmental factors leading to gully activity; and 2) the sequence of events and their 160 timing during defrosting, to better understand the potential triggers and conditioning factors for activity. The HiRISE camera is aboard NASA's MRO (Mars Reconnaissance Orbiter) spacecraft, and acquires 166 images with a spatial resolution of 0.25-1 m/pixel and three filters (RED, BLU and NIR centred on 694, 167 536 and 874 nm respectively) (McEwen et al., 2007). The CaSSIS camera is on board ESA's TGO 168 (Trace Gas Orbiter) spacecraft. CaSSIS acquires images with a resolution of ~4.6 m/pixel and four 169 possible colour filters (BLU,PAN,RED and NIR centred on 497.5,677.4,835.4 and 940.2 nm 170 respectively) (Thomas et al. 2017). 171 Given the large dimensions of our study area (68°-74°S; 345°-5°E) we divide the region into 172 "sites", with smaller "areas" defined within each site (Fig. 1). Image data available for Sisyphi Cavi 173 have a spatially discontinuous time coverage with some sites having only one or two images. We divide 174 the region in 3 sites, each containing between 4 and 20 gullies, located at: 1) 68.5°S and 1.3°E (Fig. 1B), 175 2) 70.92°S and 1.2°E (Fig. 1C) and 3) 69.4°S and 347°E (Fig. 1D). Although these sites form the 176 majority of our data analysis, we also use images located elsewhere in Sisyphi Cavi as illustrative 177 examples. A summary of the images used is provided in Supplementary Table S1. 178 To describe the timing of events in this paper, we use the solar longitude (Ls) of the first image 179 where we observe the feature/ activity appear and the Ls when we observe it for the last time. So, the disappearance of the feature occurs after the last Ls provided, but before the Ls of the next image in the 181 sequence (all sequences are visible in the Supplementary Table S1) for the same Mars Year. As 182 interannual variability in the timing of defrosting [START_REF] Piqueux | Variability 853 of the martian seasonal CO2 cap extent over eight Mars Years[END_REF] is less than the typical time gap 183 between images, we sometimes group data from different Mars years to form more complete datasets.

184

The events documented in this paper include: appearance of dark spots, appearance of dark flows, and 185 the assessment of the continuity of frost cover.

186

Assessing frost/ice cover from optical images is challenging because of the transparency of slab 187 CO2 ice in visible wavelengths and up to 25 cm of CO2 ice can accumulate before the surface is masked 188 in the visible wavelengths (Singh and Flanner, 2016). We address this by using features/patterns in 189 optical data observed by other researchers, where these researchers used spectral datasets to confirm the 190 presence of frost. We infer the presence of transparent ice, by the fact that surface features such as ripples 191 and boulders can be weakly observed on the surface, and the surface shows dark spots and flows 192 indicative of the presence of slab ice (Andrieu et al. 2018;Kieffer et al., 2006;Kieffer, 2000, 193 2007; [START_REF] Piqueux | North and south subice gas flow and venting of the seasonal 857 caps of Mars: A major geomorphological agent[END_REF]. Brightening of the seasonal ice cover at a given location just 194 prior to its retreat has been noted in previous publications (e.g. Langevin et al. 2007;Calvin et al. 2017) 195 and is thought to represent break-up of the CO2 to smaller grainsizes or deposition of water ice frost cold 196 trapped by the CO2. We infer that on partially defrosted surfaces, the remaining CO2 ice acts as a cold 197 trap for water vapour which in turn presents a highly visible, bright surface frost, which contrasts with 198 the lower albedo of the defrosted surfaces. We classify images as completely defrosted when no 199 remaining bright patches can be observed on any surface. We are reasonably confident with this 200 interpretation as these images are always towards the end of spring in our time series and these images 201 which occur later than the time of retreat of the seasonal cap at this latitude as detected by other 202 researchers using thermal and/or spectral data (Brown et al., 2010;[START_REF] Piqueux | Variability 853 of the martian seasonal CO2 cap extent over eight Mars Years[END_REF].

203

We highlight that site n°1 has the best coverage, with 59 HiRISE and 11 CaSSIS images available, 204 so we focus most of our analysis on this site. As Supplementary Table S1 shows, there is good temporal 205 sampling of this region spanning from mid-winter to summer (from Ls 158.4° to Ls 349.5°). However, 206 the lack of images from the middle of summer to the start of winter (from Ls 349.5° to 158.4°) prevented 207 us from studying the emplacement of the seasonal frost. We further divide the site n°1 into several areas, 208 (A to D, Fig. 1B) to distinguish different parts with different hillslope orientations. In this section, we only report results from site n°1 (68.5°S and 1.3°E, Fig. 1B) due to the better multi-213 temporal image coverage (Supplementary Table S1), unless otherwise indicated. S1.

Defrosting patterns of gullies

251

Here, we report on the frost occurrence and distribution within gullies, including the alcoves, channels, 252 and aprons, during winter and spring in areas C and D (region 1). We observe a consistent sequence of 253 events across martian years, as shown in Supplementary Table S3: 254 -All surfaces are frosted until Ls ~220° (Figs. 2; 3AB; Supplementary Table S3), 255 -Then, alcoves begin to defrost, while areas surrounding the gullies begin to defrost starting at 256 ~Ls 230° (Supplementary Table S3). At the same time or just afterwards the channels and aprons 257 begin to defrost.

258

-The areas around gullies become completely defrosted as early as Ls 210° and as late as Ls 247° 259 (Supplementary Table S3), 260 -The alcoves are completely defrosted. Then the channel and aprons are the last to defrost at ~Ls 261 250° (Supplementary Table S3), where some patches of late persistent frost remain visible at 262 the bottom of alcoves and inside the channels (Fig. 3F),

263

-All surfaces are defrosted around Ls 240-300° i.e., between the middle of spring to the beginning 264 of summer (Fig. 2; Supplementary Table S3).

265

Within this sequence, gully alcoves defrost before channels and aprons on areas C and D (Fig. 3DE; 266 Supplementary Table S3). Yet some patches of frost persist inside the channel and at the bottom of the 267 alcove (Fig. 3F). In general, gullies defrost later than their surrounding terrain (Fig. 3DE; Supplementary 268 Table S3), with the notable exception of sandy areas, such as dunes. S1.

295

For each slope orientation, we observe the same sequence of seasonal defrosting and associated 296 features for each of the areas A, B, C and D, as already described in section 3.1.1, with the detailed time 297 intervals reported in Supplementary Table S4 and on Fig. 5. However, the onset of events in each area 298 is offset in time. For example, there is a lag of 37° solar longitude between the appearance of "bright" 299 frost in area C compared to area D (Fig. 5; Supplementary Table S4), and we observe a lag of 18° solar 300 longitude in the complete defrosting of area C compared to area D (Fig. 5; Supplementary Table S4).

We also observe differences in seasonal frost patterns within gullies (e.g., on the channel flanks) 302 that are related to the orientation of these slope facets, as shown in Fig. 6 and Supplementary Table S3 303 for areas A, B, and C in MY31 and in Figure 7 for site n°2. In summary, equator-facing alcove facets 304 start to defrost first, followed by equator-facing channel facets and parts of the surrounding terrain. In general, the timing of defrosting depends on slope orientation (Supplementary Table S4), and 310 the sequence of events and the distribution of remaining frost remains consistent across regions. (Figs. 311 4, 7; Supplementary Table S4). An equator-facing slope always defrosts before a pole-facing slope for 312 the same material type and slope angle (Figs. 6,8; Supplementary Table S5). Yet, the orientation of 313 slope facets inside an alcove or a channel causes differences in the relative timing of defrosting (Figs. S5). Finally, gully-alcoves always defrost before channels and aprons 315 regardless of gully orientation (Supplementary Tables 3, 4 and5).

Effect of surface type on defrosting 345

We next interrogate the relationship of dark spots and dark flows with surface roughness and gully 346 activity using HiRISE and CaSSIS data. We find that surface roughness at the meter scale may influence 347 the number of dark spots, and possibly dark flows present on the surface. We find that surface roughness 348 is present in the form of boulders, depositional and erosional elements including braiding; levees, lobes.

349

For this analysis we focus on the 3 sites shown in Fig. 1, located at: 1) 68.5°S and 1.3°E, 2) 70.92°S and 350 Boulders are common on slopes in the Sisyphi Cavi region (Fig. 10), sourced from a layer that appears 375 at the same elevation as gully alcoves. These boulders measure from 1 m 2 (the limit of HiRISE Contrasts in surface roughness as well as substrate composition and physical properties seem to 394 facilitate the onset of dark spots. For example, the transition between apron and hillslopes host numerous 395 dark spots (Fig. 12CD), which aids in the identification of this transition without having to zoom-in.

396

Another example where dark spots concentrate is at the boundary between a sandy surface and the 397 surrounding terrain, where they also sometimes evolve into dark flows if the terrain is sloping (Fig. In summary, we observe that the presence of metre-scale surface roughness produces a higher number 401 of dark spots and dark flows in winter and spring (Figs. 10,11 12). In Sisyphi Cavi, this roughness 402 takes the form of: 1) boulders (Fig. 10), 2) braided gully surfaces (Fig. 11) and 3) contrasts in substrate 403 properties (Fig. 12).

68.3°S, 1.3°E). A) A concentration of dark spots/dark flows visible on a boundary between bedrock and

Discussion

425

The aim of this work is to better understand the relationship between gully activity and seasonal frost 426 deposits in Sisyphi Cavi and to investigate if we can draw wider conclusions about this relationship for 427 gullies in general. We place our results into context with previous, related results from the literature. We 428 discuss the significance of our findings regarding the sequence and timing of defrosting first, and the 429 influence of gully activity on the presence of dark spots and dark flows second. Finally, we discuss the 433 [START_REF] Raack | Present-day 867 seasonal gully activity in a south polar pit (Sisyphi Cavi) on Mars[END_REF]Raack et al. ( ,2020) ) inventoried the gully activity of the Sisyphi Cavi area and showed that it occurs 434 on area C between Ls 234° and Ls 252° (Raack et al., 2020) and in area D, between Ls 218° and 226° 435 [START_REF] Raack | Present-day 867 seasonal gully activity in a south polar pit (Sisyphi Cavi) on Mars[END_REF]. Neither activity involved the erosion of material from the wall rock of the gully.

436

This timing corresponds to a period when the alcoves have already begun to defrost, when frost is 437 brighter and just before the surface becomes ice free (Fig. 5). For area D, we observe that a new and 438 relatively dark deposit overlies an apron that is covered in brighter frost in both instances of documented 439 gully activity (Fig. 14 B,C, Ls 226° and 227° respectively). In area C, the image acquired prior to the 440 change documented by Raack et al. (2020) shows that the gully is completely covered in frost at Ls 235 441 (Fig. 14A). The following image, ESP_048107_1115 shown in Figure 3 The fact that the gully alcoves defrost first in Sisyphi Cavi (Fig. 15; Supplementary Tables 3,4 464 and 5) independent of orientation means that a factor other than solar insolation controls the onset of ice 465 loss. The expected defrosting sequence starts at steep equator-facing slopes, then progresses towards 466 shallower equator-facing slopes to flat surfaces, afterwards onto shallow pole-facing slopes and finally 467 steep pole-facing slopes defrost. Making the assumption that the steepest slopes are found at the top of 468 the hillslope consistent with the topographic measurements by Raack et al. (2020), this should result in 469 the top of the pole-facing hillslope, where the alcoves are located, defrosting last. Instead we observe 470 that it is the pole-facing aprons that are the last to defrost. We think this could be a key feature that could 471 explain present-day activity in some martian gullies. From an informal survey of HiRISE images of 472 typical mid-latitude gullies taken during the final stages of defrosting, we found several other examples 473 where aprons remained frosted while the alcoves had already defrosted (Fig. 16). However, this does 474 not seem to be a universal pattern in all gullies -for example, for linear gullies in the southern 475 hemisphere [START_REF] Pasquon | Present-day formation and seasonal 836 evolution of linear dune gullies on Mars[END_REF] the channel and apron defrost before the alcove. The difference in 476 behaviour is likely related to the substrate properties. In some gully systems, including those in Sisyphi Bedrock is expected to have values higher than 1200 tiu (Edwards et al., 2009), but is likely mixed 490 with other materials in this area because it does not appear as continuous outcrop, hence we choose intermediate values of 750 and 1000 tiu for illustrative purposes. From these data we extracted the 492 solar longitude at which the surface temperature exceeds the condensation temperature of CO2 as 493 shown in Fig. 13. From these data it can be seen for a constant thermal inertia the expected defrosting 494 sequence starts at steep equator-facing slopes, then progresses towards shallower equator-facing slopes 495 to flat surfaces, afterwards onto shallow pole-facing slopes and finally steep pole-facing slopes defrost 496 last. Assuming aprons have a thermal inertia of 250 tiu and that alcoves are generally steeper than 30°, 497 a pole-facing alcove with a thermal inertia of 750 tiu would defrost at the same time as a pole-facing 498 apron. In contrast, a pole-facing alcove with a thermal inertia of 1000 tiu would defrost nearly 20° Ls 499 before the pole-facing apron. Therefore, we infer the alcoves likely have a thermal inertia between 500 these two extremes to explain the observations. The elevated thermal inertia of the gully alcoves with 501 respect to gully fans could be due to a number of factors including, higher near-surface ice content, 502 higher rockiness, larger grainsizes, or lower dustiness. , 2012;Pasquon et al., 2019a). For the large sinuous gully on the Matara dunefield (49.5°S; 34.7°E), 508 material from the alcove is mobilised over a frosted channel and apron (Fig. 14F; Dundas et al., 2012; 509 Pasquon et al., 2019b). New gully deposits are usually not first imaged/detected during the defrosting 510 season, and therefore we do not know if they were emplaced while the aprons were covered in frost. 511 However, other observations suggest that the activity occurs primarily during the defrosting season 512 (Dundas et al. 2019), meaning it is not unreasonable to assume that the deposits are commonly mobilised 513 over a frosted surface. The only notable exception is for linear gullies, where frost is still present in the 514 alcoves (generally) when activity occurs (Fig. 14E; Dinwiddie and Titus 2021;Jouannic et al., 2012;515 2019;[START_REF] Pasquon | Present-day formation and seasonal 836 evolution of linear dune gullies on Mars[END_REF]. We note that for gullies on sand dunes, discrete scars in the sandy alcoves 516 are often associated with activity (e.g., Pasquon et al., 2019b), yet in gullies with bedrock alcoves, none 517 of the observed activities had detectable erosion of the wall rock in the alcoves (Dundas et al. 2012;518 2019). This lack of erosion, could mean that CO2 related processes are not responsible for the formation 519 of the whole gully landform or could simply reflect that the temporal baseline of observation is not long 520 enough to detect erosion into these presumably more durable materials (Levin et al. 2022).

Dark spots and flows as an indicator of recent gully activity

537

We observe that boulders, rough surfaces inside channels and on the aprons of gullies, and contrasts in 538 surface type (e.g. the edge of a dune field) are preferential locations for dark spots (and sometimes dark 539 flows) . Boulders have been noted previously as initiation points for dark spots and fans 540 (Thomas et al. 2010). In this context, our observed strong spatial correlation between the appearance of 541 dark spots/flows and surface roughness could be explained by the following hypotheses: 542 1) Surface roughness, in particular non-homogenous thermal inertia, should cause spatial 543 heterogeneities in the temperature field of the substrate and thus also in the sublimation of the 544 ice layer. More gas may be produced on the sun-facing side of a boulder, for example, causing 545 preferential pressure build-up at that location, as warmer surfaces defrost faster. 546 2) Surface roughness (e.g. boulders or levees), in particular gradients in normal stresses caused by 547 variable overburden thicknesses or densities, could cause bending moments and mechanical 548 weakness in the overlying CO2 ice slab. This would pre-condition that location for failure when 549 the gas pressure builds up. 550 3) Both of the aforementioned factors could be at play.

551

The fact that surface roughness promotes more numerous dark spots and flows can be combined with 552 the fact that recently active gullies tend to have rougher surfaces (Dundas et al. 2019). For this reason 553 they could assist in detecting recently active sites without the need for repeat imaging. Repeat images 554 could then be targeted at this zone as active sites are often repeatedly active.

555

We now interrogate what role is played by the dark/spots flows in gully activity. Surface 556 modification events in gullies in Sisyphi Cavi are less frequent compared to gullies on sand dunes or 557 sandy surfaces on Mars, which are generally active annually (e.g. Dundas et al., 2012;2015;2020; 558 Pasquon et al., 2019b). Activity of gullies on dunes is accompanied by a similar sequence of defrosting 559 patterns to those we report for Sisyphi Cavi, including the appearance of dark spots followed by their 560 transformation into dark flows as the surface warms (Jouannic et al., 2012;2019). The activity of gullies 561 occurs generally during the final stages of defrosting. The frequency of activity on Sisyphi Cavi is 562 comparable and consistent with gullies on non-sandy surfaces e.g., crater walls (Dundas et al., 2019), 563 but in contrast observations of dark spots and flows are rather rare in these systems. For both areas 564 studied in detail in Sisyphi Cavi (areas C and D at 68°S; 1.3°E) only 2 out of 33 gullies are active 565 between MY28 and MY35 [START_REF] Raack | Present-day 867 seasonal gully activity in a south polar pit (Sisyphi Cavi) on Mars[END_REF]2020). One gully on area C is active and just once over 566 a period of 8 MY. Only one gully is active in area D in MYs 29 and 31. So, material movement in these 567 two areas seems to be relatively sparse. The disconnect between the widespread presence and annual 568 occurrence of dark spots and dark flows and the low spatial and temporal frequency of activity in gullies 569 might suggests that dark spots and dark flows are not a primary agent in triggering gully activity. .

Conclusion

In this paper we explored the process of defrosting in gully-incised slopes in Sisyphi Cavi. We explored 573 the sequence, timing, and duration of events that occur during seasonal defrost, and their connection to 574 gully and dark spot/dark flow activity. We find that the general timing and characteristics of the seasonal 575 defrosting patterns in Sisyphi Cavi are very consistent from year to year, and similar to the general trend 576 observed at a global scale for the seasonal frost deposits. Our observations show that the absolute timing 577 of the different stages of the defrosting sequence is influenced by the overall orientation of the host 578 hillslope, with equator-facing slopes generally defrosting earlier than the pole-facing ones. Gully alcoves 579 defrost first before channels and aprons and independently of orientation. This is unexpected because 580 based on incoming solar radiation the defrosting should progress from the top of equator-facing slope 581 down towards the flat floor and up the pole-facing slope, terminating at the top. We argue that this could 582 be due to the dominant effect of contrasting substrates between alcove (rocky materials) and aprons 583 (finer unconsolidated materials) over the role of solar insolation and orientation during the early stages 584 of defrost. We also observe that gullies defrost later than their surrounding terrain, with some frost 585 patches remaining in the interior of channels and alcoves at the end of defrosting, likely due to 586 shadowing.

587

We also find that dark spots and flows concentrate in areas of increased surface roughness. This 588 suggests that gully activity, which creates surface roughness via contrasts in material types, deposition 589 of boulders levees and lobes and erosion to form braiding, can facilitate the appearance of dark spots 590 and dark flows over the affected area the following winter. We therefore infer that dark spots and dark 591 flows could be used to help to detect recently active gullies on Mars without repeat imaging, but they 592 themselves are not a precursor to gully activity. 593 Finally, we note that a prerequisite for gully activity in Sisyphi Cavi is a frosted apron surface. We show 594 that such a configuration is found in gully-sites elsewhere on Mars, raising the possibility that this is a 595 common precursor for gully activity, where seasonal frost is present. We note that this type of activity 596 is not associated with detectable erosion of the wall rock in the alcoves, which could mean erosion by 597 CO2 related process is too small to be detectable in these durable materials or that another process is 598 responsible for the initial formation of gullies. 599 Tanaka, K. L., and D. H. Scott, (1987), Geologic map of the polar regions of Mars, In :USGS Map I-897 1802C 898 899 Thomas, N., Cremonese, G., Ziethe, R., Gerber, M., Brändli, M., Bruno, G., Erismann, M., Gambicorti, 900 L., Gerber, T., Ghose, K., Gruber, M., Gubler,P.,Mischler,H.,Jost,J.,Piazza,D.,Pommerol,A.,901 Rieder, M., Roloff, V., Servonet,A.,Trottmann,W.,Uthaicharoenpong,T.,Zimmermann,C.,Vernani,902 D.,Johnson,M.,Pelò,E.,Weigel,T.,Viertl,J.,De Roux,N.,Lochmatter,P.,Sutter,G.,Casciello,A.,903 Hausner, T., Ficai Veltroni, I., Da Deppo, V., Orleanski, P., Nowosielski, W., Zawistowski, T., Szalai, 904 S., Sodor, B., Tulyakov, S., Troznai, G., Banaskiewicz, M., Bridges, J.C., Byrne, S., Debei, S., El-905 Maarry, M.R., Hauber,E.,Hansen,C.J.,Ivanov,A.,Keszthelyi,L.,Kirk,R.,Kuzmin,R.,Mangold,N.,906 Marinangeli, L., Markiewicz, W.J., Massironi, M., McEwen, A.S., Okubo, C., Tornabene, L.L., Wajer, 907 P., Wray, J.J. ( 2017

Fig. 1 :

 1 Fig. 1: Map of Sisyphi Cavi on Mars: A) overview images of the 3 sites studied in this paper (Mars

  study on the analysis of HiRISE (High Resolution Imaging Science Experiment; McEwen 164 et al., 2007) and CaSSIS (Colour and Stereo Surface Imaging System; Thomas et al., 2017) image data.

  165

Fig. 2 :

 2 Fig. 2: Defrosting timing at site n°1, area C (68.5°S and 1.25°E see Fig. 1AB) for martian years 28 to

Fig. 3 :

 3 Fig. 3: Typical seasonal frost evolution in Sisyphi Cavi for site n°1, area C (68.5°S; 1.3°E)A) Dark spots

Fig. 4 :

 4 Fig. 4: Influence of gully orientation on seasonal frost deposits in Sisyphi Cavi (site n°1: 68.5°S; 1.3°E).

  284

  7). For site n°1, area A has a slope orientation of 80°-85°N, area B: 95°-105°N, area C: 130°-140°N, 285 and area D: 0° -10°N (Fig.1B). For site n°2 (Fig.7), we divide the gullies observed into two groups 286 according to their slope orientation. Slopes in gully group 2A are oriented 225° with respect to north, 287 on average, whereas group 2B has an orientation of 200°.

Fig. 5 :

 5 Fig. 5: Defrosting sequence for areas A, B, C and D which have different orientations -site n°1 (68.5°S;

  305Equator-facing alcove facets can be completely defrosted while pole-facing ones are only starting to 306 defrost. On the contrary, pole-facing alcove and channel facets are the last to defrost. Slight east (or 307 west) deviations on an equator-facing slope facet delay initial and complete defrosting, and conversely 308 advance pole-facing slope facet defrosting.309

Fig. 6 :

 6 Fig. 6: Seasonal frost distribution inside gullies for site n°1 (68.5°S, 1.3°E), areas B and C: A) only

Fig. 7 :

 7 Fig. 7: Influence of slope orientation on the seasonal frost distribution at site n°3 (69.4°S, 347°E). The

  Fig. 10: Relation between boulders and dark spots/dark flows for site n°2 (70.7°S, 1.5°E). A) The west

  398

  Fig. 11: Relation between gully morphologies (alcove, channel, apron) and dark spots/dark flows. A)

  419 a sandy substrate: HiRISE image ESP_046116_1115, Ls 159.9°. B) Zoom in on part of panel A with 420 annotations and arrows pointing to aligned dark spots. C) The edge of gully aprons have a concentration 421 of dark spots. HiRISE image ESP_028802_1115, Ls 173.3°. D) Annotated zoom in on panel C with the 422 edge of the apron outlined by arrows. Image credit: NASA/JPL/UofA. Note that each image was 423 individually contrast-stretched.

  424

  430 possible factors leading to gully activity and the possible effects of inhomogeneous frost distribution on 431 gully morphology.

  Fig. 13:Solar longitude of when defrosting of CO2 occurs on slopes with different angles, where negative

Fig. 14 :

 14 Fig. 14: Distribution of frost on gullies just before or just after gully activity: A) Site n°1, 68.5°S; 1.3°E,

477

  Cavi the alcoves are comprised of rough rocky materials, whereas the aprons are presumably finer 478 unconsolidated materials (as supported by studies of the thermal inertia of gully systems -Harrison et 479 al., 2019; Khuller et al. 2021), so there are different materials on the same slope. Dune gullies in contrast 480 have rather uniform substrate properties throughout, so follow the expected defrosting pattern (i.e. the 481 steepest pole-facing slopes, defrost last). 482 In order to investigate this further we used surface temperature data from the 1D climate 483 model runs presented in Conway et al. (2018). We extracted half-hourly surface temperature data for 484 present day obliquity and position of perihelion for surface thermal inertia values of 250, 750 and 485 1000 Jm-2K-1s-0.5 (referred to as thermal inertia units or tiu) and slopes of 0-35° in the north-and 486 south-facing orientations for 72°S. Thermal Emission Spectrometer (TES) data for the region suggests 487 the average surface thermal inertia varies between 130 and 250 tiu. However, the TES footprint is 488 >2 km, hence cannot capture local variations on the hillslopes which have a smaller spatial extent.

  489

503

  Other examples of gullies where mobilised material superposes a frosted apron exists. Dundas 504 et al. (2015) observed dark flows over a frosted surface inside two gullies located at 37.4°S; 229°E; and 505 in the northern hemisphere at 58.2°N and 89.7°E. On the dunes of Kaiser crater (47.2°S; 19.5°E), a gully 506 shows an apron covered in frost just before the new gully deposits are observed (Fig. 14D; Dundas et 507 al.

Fig. 15 :Fig. 16 :

 1516 Fig. 15: Examples with various slope orientations from Sisyphi Cavi where the aprons remain frosted

  536

  570 571

  ), The Colour and Stereo Surface Imaging System (CaSSIS) for the ExoMars Trace 908 Gas Orbiter. Space Science Reviews 212, 1897-1944. https://doi.org/10.1007/s11214-017-0421-1 909 910 Thomas, N., Hansen, C.J., Portyankina, G., Russell, P.S. (2010), HiRISE observations of gas 911 sublimation-driven activity in Mars' southern polar regions: II. Surficial deposits and their origins. 912 Icarus 205, 296-310. https://doi.org/10.1016/j.icarus.2009.05.030 913 914 Treiman, A. H. (2003), Geologic settings of Martian gullies: Implications for their origins, Journal of 915 Geophysical Research: Planets, 108(E4). 916 917 Védie, E., F. Costard, M. Font, and J. Lagarde (2008), Laboratory simulations of Martian gullies on 918 sand dunes, Geophysical Research Letters, 35(21). 919 920 Vincendon, M. (2015), Identification of Mars gully activity types associated with ice composition, 921 Journal of Geophysical Research: Planets, 120(11), 1859-1879.

Acknowledgments

 S1: CaSSIS and HiRISE images used for this study for the 3 sites 1) 68.5°S and 931 1.3°E (Fig. 1B), 2) 70.92°S and 1.2°E (Fig. 1C) and 3) 69.4°S and 347°E (Fig. 1D). x = image covers the 932 area/site indicated. Ls = Solar Longitude. MY= martian year. S2: Frost evolution for areas C and D of site n°1: 68.5°S and 1.3°E (Fig. 1B).

Supplementary Table


Sotiris Bampitzaris

Jane Echinard

Gilles Baron

Cesar Escobar- Claros

Magali Brunet

Luc Robbiola

Elodie Guilminot
email: elodie.guilminot@loire-atlantique.frconservaçãodeumasecçãodeasa

Palavras-Chave

Conservation of a WWII Supermarine Spitfire section wing EVE PAILLAUX 1

Keywords:

This study focuses on a WWII Supermarine Spitfire aircraft section wing conservation, salvaged from the seabed in 1988 and stored in a private garden. The object was in poor condition so decisions on the conservation, preservation or removal of parts of the original elements were needed. Firstly, the wing fragment was studied to determine its heritage interest and to assess its historical, industrial, technical and scientific values. This object will become a memorial to the pilot who died during the mission, in his hometown. The conservation project aims to find the best solution to both conservation priorities and exhibition needs. Based on cleaning tests on corroded, painted aluminium alloys carried out within the PROCRAFT Project, a treatment was determined. This case serves as an example of how the results of a European project can be implemented in a conservation-restoration process and how industrial conservation theory can be applied to archaeological artefacts.

Introduction -the PROCRAFT project

The Second World War (WWII) is often considered to be the golden age of military aviation, with aircraft symbolizing modernity and power. They played an important role in legendary battles, such as the Battle of Britain and D-Day, which involved around 14,000 planes (fighters, bombers and transport aircraft). During this period, the USA built over 250,000 aircraft, while the other countries produced no fewer than 500,000 planes. Most of these aircraft were either lost in combat or disappeared during war missions. Many remains can be found across Europe, both on land and at sea. At the end of WWII, some of the remaining aircraft were used in other conflicts, but the majority of the machines were scrapped, melted down, or left to corrode. Thus, for example, out of the 12,731 specimens of "B17 Flying Fortress" only 20 have survived to the present day. This represents 0.15 % of one of the most widespread models, while other types of aircraft were practically all destroyed [START_REF] Berutti | Cieli d'acqua -I relitti aeronautici del secondo conflitto mondiale nei mari del mondo[END_REF]. In France, not a single example of the two most produced French WWII aircraft, the Morane-Saulnier 406 and the Potez 63, has been preserved [START_REF] Fournier | La protection des aéronefs au titre des monuments historiques[END_REF].

WWII aircraft heritage has an undeniable historical and emotional value for Europeans, but these archaelogicals remains have only recently officially entered the field of archaeology and cultural heritage conservation. Their presence in national museums is limited. They are often cared for by volunteers and volunteer-led associations.

The discovery of an airplane wreck is challenging from several points of view: its composition and materials, its history, its legal statutes and its size and condition.

To meet these challenges, Arc'Antique laboratory has collaborated since 2020 with four main partners: CEMES -Center for Material Elaboration and Structural Studies, University of Bologna, University of Ferrara and Czech Technical University in Prague, and 21 associated partners in the PROCRAFT project (PROtection and Conservation-Restauration of Heritage aircrAFT). The last ones include scientists, State representatives, conservators, academics, museums, associations, from Canada, Greece, Sweden, Italy, Czech Republic and France. They represent all the various actors in this heritage chain benefiting from their joint expertise and capabilities.

The goal of the PROCRAFT is to create innovative procedures and solutions for each key step in aircraft conservation:  Tailored conservation-restoration techniques;  Smart coatings for outdoor protection respecting the requirements for safeguarding cultural heritage;

 Innovative solutions for preventive conservation in confined or semi-confined environments;

 Guidelines for aluminium (Al) alloys conservation for volunteer groups.

The results of this project will:

 Enhance and share knowledge about the conservation of WWII aircraft, focusing particularly on the conservation of Al alloy components;

 Contribute to the preservation of aircraft heritage;  Promote dissemination and presentation of our work to the public.

Project phases (WorkPackage -WP) (Figure 1) within the PROCRAFT aim to:

 Document constituent materials and related alterations to aircraft wrecks from six nations involved in WWII (WP 2).

 Carry out chemical and mechanical cleaning tests depending on the corrosion layer and composition (WP 3).

 Deliver surface protection studies for Al alloys (WP 4).  Understand the sensitivity of alloys according to their composition and state of degradation, characterize galvanic corrosion and active corrosion processes to implement suitable treatment (WP5).

 Define guidelines for preventive conservation of aircraft wrecks (WP 6).  Ensure public awareness and dissemination of information gained from the project (WP 7). Within the framework of the PROCRAFT, one of our associate partners, the Department of Underwater Archaeological Research in France (DRASSM), entrusted us with a wing fragment from a Spitfire Mk VII MB887 to carry out tests for WP purposes. The Spitfire wing fragment encompasses the whole range of challenges involved in conservation and long-term preservation of aircraft heritage wrecks: 1) large surface with remnants of original paint, 2) varying states of degradation and corrosion aspects, 3) inaccessible parts, 4) no climatecontrolled storage environment.

Due to its historical and technical value, we decided to use the results of WPs 3 and 5 to manage a global conservation project centred on the Spitfire fragment. This paper focuses on the direct application of the first PROCRAFT results for the conservation-restoration of this WWII Supermarine Spitfire aircraft wing fragment. The broad outlines of WP 3 results are presented but details outcomes are not shared.

Firstly, this study set out to assess the aircraft's heritage value and to adapt our archaeological conservation-restoration processes accordingly. After a complete diagnostic, parts that could be saved were selected. Stabilization treatments were assessed, and cleaning tests were performed to determine suitable treatment conditions for a damaged, painted Al alloy. This paper presents the different actions carried out to ensure the conservation of the Spitfire wing as an example of collaboration and joint decision-making process between various stakeholders including volunteers, conservators, and state representatives.

Supermarine Spitfire aircraft: Historical and technical studies

The WWII Supermarine Spitfire aircraft wing was salvaged in 1988 from the seabed less than three miles off the French coast in the English Channel. The fragment represents the leading edge, the front part of a "universal type" (or Type C) starboard wing. The machine guns are missing but some landing gear parts were still attached. The object was saved from destruction by a volunteer who kept it for over 30 years in his garden. Local authorities, supported by the "Association Bretonne du Souvenir Aérien 39-45" (ABSA 39-45 association) and the DRASSM, responsible for the object's conservation, decided to restore the wing fragment and exhibit it as a memorial in honour of the pilot who gave his life to liberate France.

The Spitfire is an iconic WWII fighter aircraft which played a pivotal role in the war in the United Kingdom air defense. It was also used as a fighter-bomber to attack targets in Europe, as well as in North Africa, the Mediterranean and the Middle East. On 1st June 1944, during Operation Rhubarb to neutralize a cargo train in France, Officer James Atkinson, a 22 year-old Australian pilot, was reported missing at sea with his plane, the MB887. Forty-four years later, off the coast of Saint Brieuc, a French fisherman recovered the wing fragment, which was authenticated by its identification plate.

The Spitfire was continuously produced in large quantities throughout WWII and underwent many modifications to improve its performance [3]. The Spitfire Mk VII was developed specifically for high altitude flying, and only 140 were produced. One of its innovations was the presence of two symmetrical radiators under the wings for the supercharger and oil cooler. This Spitfire model also had an internal fuel tank in the wings for high-altitude climbs. The tank is still present on the recovered wing fragment (Figure 2) and comes from a relatively uncommon Spitfire model. A complete example is exhibited at the Smithsonian's National Air and Space Museum, but this particular plane never took part in active combat, being used only as an evaluation aircraft. The wing fragment has great authentic historical value and symbolizes the memory of the fallen Australian pilot. It is of significant value from several points of view:  Historical value: representative of an iconic WWII aircraft, the object is a poignant symbol of the deadly air battles of the time.

 Scientific and technical value: the object is one of a series of only 140 and features a major fuel tank innovation.

 Social value and authenticity: the object is directly linked to the death of the young Australian pilot James Atkinson (bullet impact marks are visible on the wing), and could be a fitting memorial to him, highlighted with explanations on the historical context.

 Aesthetic value: despite deterioration due to its long exposure to seawater, the object can be easily interpreted and its function understood.

Materials and methods

SEM-EDS analysis

To determine elemental composition, small-sized samples were cut from the object, and were embedded in epoxy resin, polished with 800-000 SiC grade paper and finally polished by cloth with diamond paste from 3 μm down to 1 μm. Scanning electron microscopy (SEM) JEOL JSM 5800LV, operated at 20 kV and coupled with Energy Dispersive X-Ray Spectroscopy (EDS), was used to analyze the elemental composition. EDS acquisition was performed on six areas of 500 × 500 μm² with a minimum of 100,000 counts on each spectrum.

Gel preparation

Agar gel

This is a gelatinous agent consisting of polysaccharides that come from a species of red algae. It is compatible for use in solutions with a pH between 5-8. AgarArt (supplied by CTS) was used as 3 wt % AgarArt. The treatment solution was heated to 50 °C before slowly stirring in the Agar Art powder. It was then heated to 90 °C until the mixture became homogeneous and translucent. After cooling for 24 hours, the gel was heated a second time to 90 °C. For spray application, the gel was inserted into a spray gun tank at 70 °C. The gun used was a Wagner W450 wall sprayer model.

Xanthan gum

This is a gelatinous agent consisting of polysaccharides, obtained by the fermentation of simple sugars using bacteria. Both products are basically Xanthan gum but Vanzan (supplied by CTS) is industrially modified. It is compatible for use in solutions with extreme pH. Xanthan Gum (supplied by Kremer) was used as a 2 wt % whilst Vanzan was used as a 5 wt %. The treatment solution was mixed with Xanthan powder at room temperature and the gel was used 24 hours after preparation.

Examination and diagnosis

The object is fabricated from: Al alloys, ferrous alloys, elastomers, synthetic materials, paint (remains). The wing is mainly fabricated from Al alloy (Al-Cu) that is highly sensitive to corrosion. SEM-EDS analysis determined that most parts are made of Duralumin with an elemental composition of Al (93.3 %), copper -Cu (4.2 %), magnesium -Mg (0.9 %), manganese -Mn (0.65 %), silicon -Si (0.5 %), iron -Fe (0.4 %) and traces (nickel -Ni, zinc -Zn, titanium -Ti, chromium -Cr). Records [3] show that Duralumin parts were protected by Al cladding (pure Al hot-rolled on both sides of the alloy, to provide more resistance to corrosion) and painting. However, SEM analysis of samples taken from different areas of the wing did reveal cladding only on the stringers. Residues of the original paint are still visible on the upper skin with an average thickness of 20 µm.

After 44 years on the seabed, the wing fragment was stored for a further 34 years in a private garden (Figure 3). The plane's history has resulted in a wide range of visible deterioration; mechanical degradation caused by the combat and crash (Figure 4a), deterioration due to seawater (Figure 4b-c), and the presence of undergrowth during the period of outdoor storage in the garden (Figure 4d). In our conservation process we undertook a full condition assessment including of the mechanical and electro-chemical deterioration. The main observations are summarized in Table 1. The most severe mechanical degradation of the aircraft probably dates from the time of the crash. The fisherman recovered only the front part of the starboard wing which accounts for no more than 30 % of the total area of a Spitfire wing. Significant degradation also took place during the period it was in the sea and stored outdoors. The internal structure had undergone extensive damage. There are major differences of state between the interior and exterior parts. The internal wing structure was in poor condition; the front spar, stringers and ribs no longer provided adequate mechanical strength due to major exfoliation and pitting corrosion. The corrosion had buckled and lifted the Al sheet (Figure 5). The ribs near the tank were in good condition, while others were in very heterogeneous condition (poor to very poor). All of the ribs displayed localized corrosion, some of which had deteriorated into exfoliation. The location of the different degradations, which were classified into four categories: localized corrosion with pitting, original paint residues, rust stains and missing rivets (Figure 6). The skin (top and bottom) was in good condition overall with traces of paint still visible (Figure 3). Paint remains were abraded and poorly preserved. Localized mechanical surface alterations were visible but did not impact overall legibility. Mechanical damage due to bullet holes was visible on the side of the location of the machine guns. Most of the rivets that attached the ribs to the wing skin were missing. These Al-Cu alloy rivets are richer in Mg than Duralumin and are more susceptible to corrosion. The tank and elastomer parts do not seem to have deteriorated. The Fe bolts had resisted better and were still present. On the other hand, the presence of Fe elements had caused galvanic corrosion by accelerating the degradation of the Al alloys. The Fe elements were slightly corroded, and Fe corrosion products had caused rust stains on the surface of the wing.

Duralumin is highly sensitive [START_REF] Vargel | Corrosion of aluminum[END_REF] when in contact with chlorides, developing pitting and intergranular corrosion aggravated by the presence of intermetallic precipitates (Figure 4b).

Records [3] show that Duralumin parts of the Spitfire were usually protected by Al cladding (pure Al hot-rolled on both sides of the alloy, to provide more resistance to corrosion) and painting. However, SEM analysis of samples taken from different areas of the wing did not reveal any cladding. During the period of storage in the garden, the wing fragment was under a plastic tarp, surrounded by vegetation which generated humid conditions resulting in increased Al alloy corrosion. Localized corrosion had become generalized in some areas, especially at the level of the internal structure. Al alloys are highly sensitive to variations in relative humidity which produces mechanical stress and enhanced mechanical deterioration. The disappearance of the rivets due to corrosion had worsened the mechanical condition of various elements which were no longer physically held together.

Pitting and intergranular corrosion is common in archaeological Al alloy wrecks. In the case of a DC-3 aircraft wreck [START_REF] Tengnér | The preservation of a marine archaeological DC-3 aircraft[END_REF], variable degradations were observed: some parts were very degraded, while others looked as if they had just come out of the factory. For wrecks salvaged from the sea, the first treatments to be developed were inspired by the stabilization of ferrous materials [START_REF] Macleod | Conservation of aluminum alloys corroded under water: from in-situ conservation through storage to exhibition[END_REF][START_REF] Degrigny | The conservation of historical marine, terrestrial and industrial aluminium objects: a review[END_REF][START_REF] Bailey | A critical review of the 1996 conservation treatment of a World War II aircraft carried out at the Australian war memorial[END_REF][START_REF] Macleod | Stabilization of corroded aluminium[END_REF][START_REF] Degrigny | Stabilisation de moteurs d'avions immergés[END_REF] involving chemical or electrochemical rinsing to remove chlorides. However, a study on the effectiveness of these treatments showed that very few chlorides remained present in the Al alloys [START_REF] Tissier | Characterization of two propeller blades from submerged WWII-era wrecks[END_REF] when the object is no longer in contact with a chloride environment. In fact, the absence of compounds containing chlorine and aluminium is probably due to the high solubility of these compounds [START_REF] Graedel | Corrosion mechanisms for aluminum exposed to the atmosphere[END_REF]. The chloride extraction treatment step is therefore not considered necessary, but additional conservation treatments remain essential to ensure the preservation of this type of Al alloy wreck.

The wing skin was in relatively good condition but required cleaning. Due to corrosion of the Duralumin, internal structure parts were in really poor condition, particularly the front spar and stringers (Figure 2). The front spar is the major structural element and this required urgent remedial conservation treatment (Figure 4d) as it had developed extensive exfoliation. Stringers needed consolidation, while some ribs that were too severely degraded had to be removed (about 10 %).

Conservation objectives

By taking into account the object's history and background, conservation state, diagnosis results and exhibition expectations, we determined the following conservation objectives:  All elements that improve the overall understanding of the functional and technical characteristics must be preserved and consolidated, whereas corrosion products or parts that are not relevant to the object's original function should be removed.

 Because of its archaeological origin and the fact that remnants of paint subsist in some areas, the final surface appearance will be homogenized rather than standardized.

 Non-adherent exogenous deposits will be removed as well as marine concretions. Surface corrosion products, mostly hydrated aluminium oxide, will be cleaned or removed.

 An exhibition will take place in the town hall close to where the wreck was found. It is an indoor environment with unstable environmental conditions. Sufficient surface protection will be provided to ensure the stability of the metal and compounds.

Treatment has been carried out for a three-month period using the results of tests from the WP 3. The most suitable treatment for the Spitfire wing has been chosen to achieve conservation objectives.

PROCRAFT: results of previous cleaning tests

As the wing surface presented varying surface conditions, the specific treatment protocol had to be determined, and therefore the results of previous testing were considered. WP 3 research established cleaning protocols by carrying out tests on metal surfaces, corroded surfaces and painted surfaces from authentic WWII aircraft wrecks. The objects included a propeller blade from an unidentified aircraft found in the Bay of Brest, a stiffener fragment from a Dewoitine D338, abandoned after WWII near Marignane airport (France), and a plate fragment from a Messerschmitt Bf109, which crashed during WWII at Le Rheu (west of Rennes, France). Assessment and description of corrosion layers were carried out on the different objects before evaluating different cleaning methods. In these cleaning tests, mechanical techniques and chemical treatments were compared. The mechanical techniques, most widely used by conservators, such as brushing with or without water, using a scalpel, micromotor and blasting were tested. Results are shown in Table 2.

Chemical treatments were applied using Agar or Xanthan gels. Many of the treatment solutions were selected based on previous studies on Al alloy treatments. All chemical treatment solutions are listed in Table 3. On this type of surface, rinsing only took place when all the other treatments had been carried out, and consisted of very light rubbing with a cotton swab soaked in deionised water, until no further residue was deposited on the swabs. Agar or Xanthan [START_REF] Macleod | Conservation of aluminum alloys corroded under water: from in-situ conservation through storage to exhibition[END_REF][START_REF] Macleod | The effects of chloride ions on the corrosion aluminium alloys used in the construction of Australia II[END_REF] EDTA cleaned off most of the surface deposits and removed corrosion products, the surface was clean after a minimum of 6 ten-minute gel applications. EDTA solution was the most effective but can slightly damage paint. Sodium Metalicate (2 % w/v) 9 Xanthan [START_REF] Macleod | The effects of chloride ions on the corrosion aluminium alloys used in the construction of Australia II[END_REF] Sodium metasilicate had little effect on corrosion products even after 60 minutes of treatment. On the painted surface, sodium metasilicate yielded the best results so far as it removed exogenous deposits without inducing any discoloration of the painted surface Ammonium sulphate (0.125 M) + Ammonia (NH3) (0.25 M)

9.6

Xanthan [START_REF] Macleod | Stabilization of corroded aluminium[END_REF] The ammonium sulphate and ammonia mixture worked well but yielded a matte finish Ammonium sulphate with aqueous ammonia dissolved paint remains and is not recommended for painted surfaces. Cationic Resin by CTS (120H Cation)

2.9

Only with water [START_REF] Carlino | Utilisation des résines échangeuses d'ions pour le nettoyage d'objets métalliques archéologiques ou historiques[END_REF] 120H Cationic Resin also gave excellent results. However, there are limitations due to its fast reaction time and it is not suitable for larger surfaces Finally, different cleaning techniques were tested: mechanical techniques and chemical treatments applied by gel. On corroded surfaces, the best results were obtained by blasting with vegetal abrasive, or by applying EDTA gel. On painted surfaces, the best results were obtained with chemical treatments. The choice of the active agent depended on which compounds were to be removed. When the paint strongly adhered, mechanical techniques, like blasting with vegetal abrasive or brushing with water, also gave very good results.

Because objects tested in WP 3 present similar materials and corrosion issues and that they could be directly relates to the choice of Spitfire treatment, the results of previous testing were employed on the Spitfire Mk VII MB887 wing fragments. Thus, for the parts of the Spitfire wing with residues of paint, the selected treatment solution was TAC.

Treatment choices for the Spitfire Mk VII MB887 wing fragment

The first step was to clean the internal area, removing any sediment, soil, sand, concretions and vegetation still present. Micro-suction enabled most of the various deposits to be extracted. After preliminary cleaning, the object gained considerably in terms of legibility. It was then necessary to consolidate the most severely damaged parts of the structure. For those parts that were exfoliating, an initial, in-depth consolidation was made with Paraloid B44 at 15 % w/v in acetone, then a second consolidation on the surface with Paraloid B44 at 30 % w/v in acetone. The front spar was lined with fiberglass using Paraloid B72 at 30 % or 60 % w/v in acetone. It was necessary to fill some of the internal parts to stabilize all the elements and restore the overall appearance of the object. Fills were made of Paraloid B72 (at 60 % or 70 % w/v in acetone) loaded with glass microspheres and pigments. Marine concretions were removed from ferrous parts by mechanical techniques, mainly with a rotation system, micro engraver or micro chisel. On structural Al alloy parts, mechanical techniques (sandblasting) were generally sufficient. The removal of concretions was finalized with a cotton compress impregnated with nitric acid at 10 % w (pH=1) for 30 seconds, then the surface was rinsed abundantly.

For the cleaning of the wing skin, which presented paint residue, aluminium corrosion products and rust stains, a first round of chemical cleaning was employed with a solution of TAC at 5 % w/v applied by Agar gel to 3 % w/v. Compared to other chemical agents, TAC was more efficient in removing dirt and was less aggressive. The gel was sprayed hot, in line with a protocol determined for application to large surfaces [START_REF] Giordano | New methods of applying rigid agar gels: from tiny to large-scale surface areas[END_REF]. Three 20-minute applications were necessary to remove dirt and reduce rust stains (Figure 7). Cleaning of the wing skin was followed by blasting with vegetal abrasive to homogenize the surface.

The last part of the treatment which will focus on application of a protective coating and exhibition of the wing fragment is still in progress is scheduled for completion in early 2024.

In order to select the most suitable coating, we will use the results of WP5 which will specifically address coatings. The surface protection chosen for the wing will need to ensure treatment compatibility and reversibility.

The study of the exhibition system is also still in progress. The following specifications for display have been given to the stakeholders:  The object must be mounted in such a way as to reinforce the structure and enable technical interpretation of the object;

 The interior surface must be accessible to enable long-term dusting and maintenance;

 The safety of the public must be ensured and clear information provided to help them understand the object;

 It must include a memorial to the pilot James Atkinson. The historical and technical studies of the wing fragment highlight its considerable significance in terms of heritage and historical, emotional, aesthetic, scientific and technical value. The conservation project has also enabled the development of innovative cleaning techniques for Al alloys. Established mechanical cleaning techniques were coupled with chemical cleaning. The next conservation step for the preservation of the wing fragment is the surface protection of the aluminum alloy components. Tests will be carried out in the context of the PROCRAFT to select the most effective protection. The object will then be exhibited locally in Britanny (France) as a memorial to the pilot.

Figure 1 .

 1 Figure 1. Organization of project phases within PROCRAFT.

Figure 2 .

 2 Figure 2. Exploded view of the Spitfire Mk VII wing fragment.

Figure 3 .

 3 Figure 3. The Spitfire Mk VII wing fragment stored in the garden.

Figure 4 .

 4 Figure 4. Examples of deterioration observed on the wing fragment of the Spitfire Mk VII: a) bullet impact; b) SEM photo of a sample (cross-section) from the wing showing generalized corrosion with inter-and intragranular corrosion in relation to micrometric intermetallic precipitates; c) development of marine microorganisms, called "concretions"; d) presence of undergrowth inside the wing.

1 .Figure 5 .

 15 scheme The recovered fragment is colored in green on this diagram which shows a Spitfire wing in its entirety

Figure 6 .

 6 Figure 6. Location of various degradations on the wing fragment of the Spitfire Mk VII.

Figure 7 .

 7 Figure 7. Mechanical cleaning: a) before; b) after treatment; c) consolidation; and chemical cleaning with TAC 5 % w/v applied with Agar gel to 3 % w/v by spray: d) removal of the gel; e) surface before cleaning; f) surface after cleaning.

Table 2 .

 2 Mechanical techniques used and the results obtained.

	Mechanical Treatment	Impacts on clean metal surface	Impacts on corroded surface	Impacts on painted surface
	Scalpel	-Unsuitable	Suitable for local corrosion products	Suitable for local use
		-Time-consuming for large surfaces
		-Risk of scratching
	Micromotor with steel brush	-Unsuitable	Unsuitable	Unsuitable
		-Time-consuming for large surfaces
		-Risk of scratching
	Blasting with a vegetal abrasive		Most effective in removing mild
			surface corrosion, resulted in a
			homogeneous surface.
	Soft brush with deionized water			Only effective if the paint
				adheres strongly to the surface

Table 3 .

 3 Chemical treatments used and the results obtained.

	Treatment Solution	pH	Gel	References	Results
	Citric acid (0.055 M)	5.4	Agar	[13-14]	citric acid or tri ammonium citrate (TAC) solutions remove corrosion products,
					but it took at least 6 to 12 ten-minute applications of gel to achieve a clean
					surface with a shiny finish
	Tri-Ammonium Citrate	7.5	Agar	[15-16]	TAC cleaned off most of the surface deposits and removed corrosion products,
	(TAC) (5 % w/v)				but also removed the thinnest parts of the painted layer of the Messerschmitt
					object. TAC had limited effectiveness but did not modify the paint
	EthyleneDiamineTetraAcetic	8
	Acid (EDTA) (0.5 M)

Acknowledgements

The authors express their sincere gratitude to the Association ABSA 39-45 for providing information on the object and the DRASSM (Département des recherches archéologiques subaquatiques et sous-marines) for its time. We also would like to thank the Direction Régionale des Affaires Culturelles (DRAC) Pays de Loire, JPI-CH and French National Research Agency (ANR) for their financial support. And special thanks to Lisa Preud'homme for her photos.

Artefacto arqueológico

Conservation of a WWII Supermarine Spitfire section wing