Laure Bonnaud

Jérôme Coppalle

Co-regulation in Practices: The Hygiene Package in French Slaughterhouses

In Europe, the food safety policy combines the regulatory interventions of member states with private standards. The Hygiene Package, which came into force on the 1 January 2006, is characteristic of this model. The aim of our article is to show that whilst we can learn about co-regulation by examining the standards and principles on which it is based, we will gain an even better understanding by examining its actual implementation. From a sociological standpoint, we look at how the Hygiene Package was received and implemented in French slaughterhouses. (1) After setting out the structure of slaughterhouses in France and the ways in which veterinary services are organised, (2) the article looks at how inspection services have been affected by the introduction of the Hygiene Package, particularly with regard to the changes in relationships within the inspection service, between veterinarians and official auxiliaries and (3) it then examines the evolution in roles and responsibilities between veterinary services and food business operators.

'The French authorities have continued their work on implementing the 'hygiene package' in meat and dairy establishments. Official checks are carried out on the basis of a risk analysis and in a co-ordinated and consistent manner. (...) The observations which were made attested to the existence of unhygienic work processes: a whole series of stored bovine and horse carcasses showed visible signs of contamination (hairs, faecal matter); pig carcasses were poorly shaved; barrels of blood (for human consumption) contained dried scabs and dirt mixed in with the blood; in addition, the equipment used to collect pig blood was inappropriate, corroded, dirty. The gutting room was in an even more disastrous state, with manure and animal waste in a great many places, a 'Parmentière machine' 1 covered with hairs, waste and dirt. Furthermore, category 1 and 3 animal waste was stored here. The tripe butcher's clothing was disgusting and the adjoining toilet was repulsive'. Extract of the report on a food and veterinary office mission on Food hygiene in France, 2009 T he Food Veterinary Office (FVO) assesses conformity to the Food Law in the different member states, on behalf of the European Union. In 2009 its report concluded that France had properly implemented the new approach in slaughterhouses,

but it was extremely critical of the levels of hygiene in the slaughterhouses visited (FVO 2009a). Similar conclusions were drawn with regard to Italy, (FVO 2008a), Denmark (FVO 2008b) and the UK [START_REF] Fvo | European Commission, DG (SANCO)[END_REF]. How can we explain this paradox?

In Europe, the food safety policy combines the regulatory interventions of member states with private standards [START_REF] Ansell | What's the beef? The contested governance of European food safety[END_REF][START_REF] Marsden | The new regulation and governance of food. Beyond the food crisis?[END_REF]). The Hygiene Package, which came into force on 1 January 2006, is characteristic of this model, 'a hybrid of state and non-state actors' (Majone 1996;Egan 2001, p. 264). Made up of a series of regulations 2 , its purpose is to ensure food safety at all stages of the production process, from primary production up to and including sale to the final consumer, e.g., from the farm to the fork. It states that food business operators are responsible for the products that they sell and that they must enact safety procedures based on an analysis of the dangers within their premises, in accordance with Hazard Analysis and Critical Control Points (HACCP) 3 . It thus falls within the movement to extend the good practice guides, quality systems and standards, which regulate farming sectors, agrobusinesses and professional practices (Brunsson and Jacobsson 2000). As these standards are based upon expertise which is perfectly suited to a specific industrial sector [START_REF] Kerwer | Rules that many use: standards and global regulation[END_REF][START_REF] Demortain | Standardising through concepts. The power of scientific experts in international standard-setting[END_REF]), they are generally considered to be the most adequate and effective. The Hygiene Package is an example of co-regulation: 'Co-regulation combines binding legislative and regulatory action with actions taken by the actors most concerned, drawing on their practical expertise' (Commission of the European Communities, 2001, p. 21). Its effectiveness is deemed to be greater for the production of standards, their implementation and their application [START_REF] Henson | Food safety regulation: an overview of contemporary issues[END_REF][START_REF] Eijlander | Possibilities and constraints in the use of self-regulation and co-regulation in legislative policy. Experiences in the Netherlands-Lessons to be learned for the EU?[END_REF][START_REF] Martinez | Co-regulation as a possible model for food safety governance: opportunities for public-private partnerships[END_REF].

We accept that the extension and implementation of private standards vary considerably according to the country and sector (Marsden et al. 2010, p. 267). The aim of our article is to show that whilst we can learn about co-regulation by examining the standards and principles on which it is based, we will gain an even better understanding by examining its actual implementation. With this in mind, we look at how the Hygiene Package was received and implemented in French slaughterhouses. From a sociological standpoint, we describe how government officials are taking these regulations on board and how they are changing the way they work. We then examine how relations between official veterinarians and slaughterhouse workers are evolving on site. In this way we show that whilst a new approach to food safety is certainly being implemented, it is taking distinct forms which are sometimes far removed from the original theoretical intent.

Theoretical framework

In order to examine the implementation of this new approach to food safety in France, our article offers an analysis of the work carried out by veterinary inspectors in slaughterhouses. Literature on implementation [START_REF] Bardach | The implementation game: what happens after a bill becomes a law[END_REF][START_REF] Pressman | Implementation: how great expectations in Washington are dashed in Oakland 3rd edn[END_REF] and on frontline workers [START_REF] Lipsky | Street-level bureaucracy: dilemmas of the individual in public services[END_REF][START_REF] Maynard-Moody | Cops, teachers, counsellors: stories from the frontlines of public services[END_REF][START_REF] Jeannot | Les fonctionnaires travaillent-ils de plus en plus? Un double inventaire des recherches sur l'activité des agents publics[END_REF]) is a reminder that individuals out in the field are major actors of public policies, of their successes and failures. It suggests that the analysis of public action should not be confined to the examination of policymaking, but that it should strive to understand the commitment and practices of the actors at both central and local levels. In the case of regulatory policies, sociological analyses are more often than not inspired by [START_REF] Weber | Wirtschaft und Gesellschaft 1980 Edition[END_REF] sociology of law, which sees legal order not as a series of imperatives, but rather as a set of regulatory resources which are mobilised to a greater or lesser extent depending on the situation [START_REF] Lascoumes | Normes juridiques et mise en oeuvre des politiques publiques[END_REF]). Research on the regulators typically looks at the style of regulation that an inspector might personify, from an accommodating approach based on conciliation and co-operation, to a dissuasive or coercive approach which tends more towards repressive measures [START_REF] Hawkins | Environment and enforcement: regulation and the social definition of pollution[END_REF][START_REF] Reiss | Selecting strategies of social control over organizational life[END_REF]. This perspective opened the door to numerous studies of greater depth and it is now accepted that regulators successively adopt different approaches, depending on the circumstances, who they are dealing with, the strategies of the monitoring bodies and how they conceive their profession (i.e., [START_REF] Braithwhaite | An enforcement taxonomy of regulatory agencies[END_REF][START_REF] Gormley | Regulatory enforcement: accommodation and conflict in four state[END_REF][START_REF] Gormley | Regulatory enforcement styles[END_REF][START_REF] May | Making sense out of regulatory enforcement[END_REF][START_REF] May | Reconsidering styles of regulatory enforcement: patterns in Danish Agro-Environmental Inspection[END_REF]Pautz 2009a). As [START_REF] Harrison | Contested regulatory practice and the implementation of food policy: exploring the local and national interface[END_REF] point out, the study of regulatory practices at different levels raises new questions, in particular that of their integration: how can we establish a link between the diversity of the practices observed at local level and a 'single' policy at national or European level?

In this research (with the exception of Pautz 2009b), the regulatory framework in which inspectors operate is stable and unambiguous. In our case, through the Hygiene Package, the new nature of the regulation changes the role and work of the government inspectors responsible for monitoring slaughterhouses. For veterinary inspectors, it is no longer a case of just examining the animals, of carrying out laboratory tests to detect germs and parasites and of verifying food-handling hygiene, but also of ensuring that the HACCP plan defined by the slaughterer is being properly implemented. Studies on the concept of HACCP demonstrate that it is subject to varying interpretations [START_REF] Untermann | Food safety management and misinterpretation of HACCP[END_REF]. For example, Ten [START_REF] Ten Eyck | Is HACCP nothing? A disjoint constitution between inspectors, processors and consumers and the cider industry in Michigan[END_REF] show that it takes on different significances depending on the actor in the agrobusiness system: Michigan's cider inspectors, food business operators and consumers all understand it in a different way. Furthermore, not only do the tasks change, so does the value that the inspectors give them, along with the hierarchy that they establish between them: whilst meat inspection has been the core activity of inspectors since the nineteenth century, it is now just one task among many. Whilst documentary inspection was part of the red tape -of the 'dirty work' -it is now an obligatory stage of slaughterhouse inspection. With this in mind, our article looks at how the Hygiene Package is changing the inspector's occupations and their relations with slaughterhouses.

Methods

Our article is based on observations and semi-directive interviews within veterinary services in five French regions, between September 2008 and June 2009. The study took place in slaughterhouses of all sizes, both single and multi-species. Laure Bonnaud spent periods of between three days and several weeks in the inspection services of the slaughterhouses concerned. She met with all inspectors, e.g., official veterinarians and official auxiliaries, taking part in their daily work and then carrying out interviews of varying lengths (one to several hours); finally, she interviewed slaughterhouse executives (the directors of the smaller sites and both the directors and the quality managers at the larger sites). The interviews were recorded and then typed out in full in order to allow joint analysis by the authors. Our article is thus based on observations and on the analysis of 83 interviews which took place in 15 different slaughterhouses.

After setting out the structure of slaughterhouses in France and the ways in which veterinary services are organised (1) the article looks at how inspection services have been affected by the introduction of the Hygiene Package, particularly with regard to the changes in relationships within the inspection service, between veterinarians and official auxiliaries, (2) it then examines the evolution in roles and responsibilities between veterinary services and (3) food business operators.

Veterinary inspection in French slaughterhouses

A longstanding command and control approach. The case of slaughterhouses presents several characteristics which make it particularly heuristic. First and foremost, as elsewhere in Europe, the French government has been intervening to ensure the salubrity of meat since the end of the nineteenth century [START_REF] Hubscher | Les maîtres des bêtes. Les vétérinaires dans la société française (xviiie-xxe siècle)[END_REF][START_REF] Koolmees | Veterinary inspection and food hygiene in the twentieth century[END_REF][START_REF] Enticott | The changing role of veterinary expertise in the food chain[END_REF]. Veterinary inspectors are present both when the animals are delivered and on the slaughter chain, where they examine each carcass and its offal. Their objective is to use their senses and their anatomopathological knowledge to identify lesions and anomalies which show that the animal has a pathology that renders its meat and/or offal unfit for human consumption. Over the course of the twentieth century, the gradual understanding of the connections between animal diseases (trichina, tuberculosis, brucellosis, etc.) and meat which was unfit for consumption led to improved techniques and added to the expertise of veterinarians [START_REF] Hardy | Animals, disease and man. Making connections[END_REF]Stanziani 2005, pp. 223-255). Inspection gradually integrated new knowledge and tools, combining the sensorial approach with laboratory tests [START_REF] Hardy | Food, hygiene and the laboratory: a brief history of food poisoning, circa 1850-1950[END_REF][START_REF] Stanziani | From laboratory expertise to litigation: the municipal laboratory of Paris and the London inland revenue laboratory, 1870-1914. A comparative analysis[END_REF] and documentary analysis [START_REF] Torny | La traçabilité comme mode de gouvernement des hommes et des choses[END_REF]. These evermore extensive checks nevertheless fell within the traditional 'command and control' approach to food safety regulation, whereby the government enacted rules and ensured their application.

With the Hygiene Package, slaughterhouses remain subject to permanent inspection, e.g., veterinarians continuously check carcasses, just as they used to. But with regard to all other aspects (hygiene, microbiological analyses, etc.) food business operators are fully responsible for the products they sell. They must identify the dangers inherent in the operation of their establishments and come up with a plan setting out the measures they have implemented to prevent or correct said dangers, in accordance with the HACCP method. Veterinary inspectors have a dual role to play: first of all, they authorise establishments to operate by granting a permit, in accordance with the health and hygiene system proposed by the slaughterhouse; secondly, they check that the safety system thus implemented is operational. References for inspection vary, depending on the establishment, its expertise and its manufacturing procedures. In Europe, the Hygiene Package has not changed the systematic inspection of every carcass and HACCP plans coexist with this historical method of monitoring salubrity. The situation is different in the US, where in 1996 the introduction of the Pathogen Reduction Act led to the removal of the USDA's federal inspection, whose officials no longer carry out ante-or post-mortem inspections (Juska et al. 2000, p. 262).

Slaughterhouses in France.

France is one of the largest agricultural countries in the European Union. It is the leading cattle producer (1,510,000 tons in 2006 and 21 per cent of the production of the member states (Eurostat 2008), the second biggest sheep producer after Spain, and the third largest pig producer after Germany and Spain. The consequence of such a large and diverse production is that France has 262 approved slaughterhouses, 68 per cent of which are multi-species (MAAP 2010). Slaughter takes place mainly in the west of France: Brittany counts for 39 per cent of slaughter, all species taken together, whilst over half of the slaughter (52 per cent) is carried out in just two regions -Brittany and the Loire region (Figure 1). This geographical concentration goes hand in hand with an industrial concentration, with 41 slaughterhouses in the west (out of 262, e.g., 15.6 per cent) carrying out 50 per cent of slaughter in France. This concentration also means that small slaughterhouses, located all over France, coexist with very large abattoirs, essentially in Brittany. In 2007, 204 slaughterhouses were each processing less than 10,000 tons per year, with 129 of them having a capacity below 2,000 tons. The introduction of the Hygiene Package thus took place within a highly heterogeneous landscape. 'Vets'. In an ethnographic work carried out by participant observation, [START_REF] Muller | Quae)[END_REF] noted that the slaughterhouse workers called all members of the inspection teams 'vets'. Yet this term covers people with very different levels of experience, qualification and status. The staff in the veterinary services in slaughterhouses include 519 veterinarians, e.g., 26 per cent of the 2010 inspectors working in slaughterhouses [START_REF] Maap | Extrait du logiciel Sigal pour le bureau des abattoirs (Paris: Ministère de l'Alimentation[END_REF]. 74 per cent of the inspectors are thus official auxiliaries. Of these 1168 auxiliaries, 78 per cent are civil servants: they passed a competitive examination and received two years of training, both theory and practice, in a vocational school. The remaining members of the inspection teams are contract workers, most of them with fixed-term contracts -they are able to join the teams for a few weeks or months, often without any training. On the other hand, only 49 (9.5 per cent) veterinarians working in slaughterhouses are civil servants: 90.5 per cent are temporary replacements. Some work mainly for the veterinary services and have sometimes been doing so for decades, particularly those in the major abattoirs; others maintain their freelance medical and surgery activities and work from time to time as inspectors, especially when it comes to making decisions on seizure or carrying out ante-mortem inspections. Whatever their status may be, the veterinarians are considered to be in charge of the slaughterhouse inspection service: temporary official veterinarians thus have to manage inspection teams mainly composed of civil servants.

Within the inspection teams, the division of labour has always been very clearly marked. The veterinarians work office hours and never go onto the slaughter chain. The official auxiliaries inspect the meat on the chain and must therefore be present as soon as slaughter begins (between 3am and 5am). Direct observation shows that the two categories of inspector are nevertheless interdependent. Carcass inspection is largely a question of experience: the more lesions an inspector has seen, the more easily he can spot them. It is the official auxiliaries who are permanently present on the chain. Yet according to the regulations, seizure decisions must be made by a veterinarian. The latter are often less competent than their team when it comes to detecting anomalies, especially as meat inspection has a poor reputation in veterinary schools. It is therefore very important that veterinarians and official auxiliaries find ways of working together for inspection [START_REF] Bonnaud | La production de la sécurité sanitaire au quotidien: l'inspection des services vétérinaires en abattoir[END_REF]. We therefore find several factors of heterogeneousness in the implementation of meat safety policy: regulations comprise two types of standard -private and public; French slaughterhouses are very diverse, running from small para-municipal multi-specie slaughterhouses which operate just a few days a week, to those belonging to major industrial groups which work 16-24 hours a day. Finally, the inspectors themselves form a heterogeneous group whose members do not always have the same aspirations. In what ways has the Hygiene Package changed inspections?

The reception and implementation of the Hygiene Package within French veterinary services

In France, the implementation of European food safety regulations led to a reorganisation of veterinary services in slaughterhouses. The Ministry of Food and Agriculture reinforced its tools for monitoring inspections and attempted to more accurately define the role and responsibilities of government officials working in slaughterhouses.

European regulations and inspection reforms. Since the mid-1990s and parallel to the changes in European food safety regulations, French veterinary services have been working on the implementation of quality assurance in inspections, in accordance with the ISO 17020 standard: 'General criteria for the operation of various types of bodies performing inspection'. Those in favour of this decision believe certification by a third party within public administration to be a logical consequence of European directive 93/43 which encourages a quality approach in agrobusinesses (Gerster et al. 2003, p. 621). As can be seen from this speech given by the French minister of agriculture on the 27 January 1997, the private sector is seen as a model for the inspection service:

I want you to introduce quality assurance procedures in your service as rapidly as possible.

It is vital that we ensure that inspections are perfectly coherent throughout the country, but, more fundamentally, it would be somewhat paradoxical to force upon food business operators obligations that we are not able to meet ourselves.

Concretely procedures and traceability tools have been introduced. Documentary systems are now generalised, with the idea that proper work is work which is done in accordance with an instruction, with its completion being recorded in order to be able to provide any documentary evidence which might be required at a later date by consumers or the courts. At the same time, veterinarians are disputing their administrative attachment to local agriculture development departments. Their representatives are criticising these departments as being linked to the economic interests of breeders and inclined to making deals with farmers' unions. The ISO 17020 approach is thus being brandished as a proof of integrity and impartiality.

As from 2002, veterinarians became independent of agricultural development departments and quality assurance became generalised. The organisation of veterinary inspection is thus moving more towards formalisation, with a view to clarifying who does what and who is responsible for what. What are the consequences for the inspector occupation?

Formalising practices in a context of permanent inspection. Concretely, the ISO 17020 standard involves formalising inspection methods by introducing written working procedures, acquiring tools for the standardisation of acts (manuals, guidelines, grids) and implementing the traceability of acts. All inspectors have identified this formalisation of practices as representing major change in their occupation, but veterinarians and official auxiliaries do not feel the same way. For the veterinarians in charge of the inspection services, the implementation of quality assurance is seen as a positive sign of the modernisation of administration, which will allow it to achieve greater recognition. The teams, on the other hand, are concerned about how the formalisation of their activity will affect their everyday work. Indeed, official auxiliaries work on a permanent basis with the slaughterhouse workers. They therefore make an effort to maintain good working relations with them, because the latter are in a position to make their job easier (by pointing out suspicious carcasses for example) or, on the contrary, much harder, should they decide to hide problems [START_REF] Muller | Quae)[END_REF]. For official auxiliaries, the obligation to spot failings and then put them in writing makes it complicated to maintain relationships with the workers, whose cooperation is needed to carry out the inspection: 'Written reports stigmatise individual responsibilities in relation to incidents and crystallise judgments of individuals' (Reverdy 2000, p. 240). In the following extract, one official veterinarian explains the difference between his own conception and that of his team regarding the recording of inspection activity:

There's this chain foreman, but he doesn't do his job, he asks any old person to remove veal tonsils 4 , someone who knows nothing about it. So the guy told me 'Oh, no-one has ever shown me what to do'. Afterwards I talked to the official auxiliary, who said 'Hang on, I've personally told him four times how to do it'. The problem is that there is no written trace which says 'this operator is incapable of removing Specified Risk Material (SRM), I've told him four times how to do it'. That's something the auxiliary never wrote down. So joint responsibility! For each of the four times we should have been able to find a written trace in the slaughterhouse's internal log, with the auxiliary stating: 'that's four times I've explained to this operator what to do and he still can't do it properly'. But there is no trace. So our responsibility is fully engaged. So there you go! We are jointly responsible and that's something that can lead to a European stamp being withdrawn. (Official veterinarian) Rather than systematically noting operators' mistakes and sending written reports to their superiors, teams employ various tactics which allow them to note failings whilst at the same time preserving interaction. For example, they might make a joke which shows that they have noticed the operator's mistake and that they are keeping an eye on him or her, and they always prefer to explain, to demonstrate the correct way of doing things or to 'make do' with the people they are inspecting, in an approach that they describe as 'pedagogical'. In theory, such behaviour is not compatible with the introduction of quality assurance for inspections, which involves a written report for each action of inspection. A barrier is thus being built between official auxiliaries, who work alongside operators, and the veterinarians who manage the service. Here we encounter the results highlighted in the field of social politics [START_REF] Standfort | Moving beyond discretion and outcomes: examining public management from the front lines of the welfare system[END_REF]: regulations of formalisation create a rift between managers and the personnel out in the field. unacceptable incident had taken place without any reaction from the inspection service.

With the changes that have been made to the inspection process, teams are finding this type of misunderstanding to be increasingly frequent. Veterinarians share their teams' opinions on irregular situations, but they often disagree about the type of solution that is required. They favour inspections which take place in a situation of power, e.g., where their actions cannot be challenged at a regulatory or technical level. Official auxiliaries feel that they have lost all power, the latter having shifted over to the veterinarians, who decide whether or not to confirm their decisions.

We thus find a new hierarchy in inspection tasks: the professional pride of official auxiliaries had for a long time been defined by the fact that they were guardians of meat quality on behalf of consumers -they were the ones who removed carcasses unfit for the food chain and who, through their inspections, ensured that there was a satisfactory level of hygiene in the various establishments. This activity has now become marginalised, partly due to overall improvements in animal health and partly due to changes in regulations, which are now based on the procedures used by the slaughterhouses themselves. Because veterinarians are responsible for the implementation of this new approach, they tend to work less closely with their teams. Paradoxically, within this evolution, official auxiliaries have greater specific knowledge and veterinary expertise (in relation to lesions and animals) than their heads of department. The latter tend to focus on risk analysis, procedures and the engineering in agrofood establishments, skills relating to production systems and modes of organisation which are normally associated with quality control managers. In this way the Hygiene Package helps to redefine veterinary expertise, as [START_REF] Enticott | The changing role of veterinary expertise in the food chain[END_REF] have shown in other fields.

The Hygiene Package thus very significantly modifies relations within the inspection service, as well as the role and status of veterinary expertise. What is the situation regarding relations with the slaughterhouses?

Relationships between inspectors and inspecteed since the implementation of the Hygiene Package

Co-regulation supposes that each party, both State and slaughterhouses, plays a role in the new regulatory framework and fully accepts the responsibilities it entails. Given that France is characterised by the considerable heterogeneousness of its slaughterhouses, how do inspectors and meat business operators approach these new roles?

When the heterogeneous nature of slaughterhouses leads to diversity within the inspector occupation. The Hygiene Package allows food business operators greater latitude to analyse risks and develop their own procedures. However, this opportunity that the Food Law provides is not always taken up. In certain smaller establishments, there are limited resources with which to analyse risks and develop an HACCP plan, especially at the human level: trainees, quality managers with fixed-term contracts or working at multiple sites, etc. [START_REF] Worosz | Resilience in the US red meat industry: the roles of food[END_REF]): always playing catch-up, we aren't a driving force at all! (Site Director, multi-species slaughterhouse)

Faced with this situation, certain officials from the veterinary service help with the development of the HACCP plans in order to ensure a minimum level of food safety and regulatory conformity: they suggest stages for completing the work, advise on recruitment, on critical issues, document models, etc. [START_REF] Bonnaud | La production de la sécurité sanitaire au quotidien: l'inspection des services vétérinaires en abattoir[END_REF]. Reasoning in a similar manner to certain researchers [START_REF] Delind | Safe at any scale? Food scares, food regulation, and scaled alternatives[END_REF], inspectors feel that the risks are not the same for all establishments and that smaller structures must be preserved because they deal with specific production types (quality labels and short circuits). In so doing, they see themselves more as educators than as enforcers [START_REF] May | At the regulatory front lines: inspectors' enforcement styles and regulatory compliance[END_REF].

On the other hand, meat business operators who have taken the new regulations fully on board want the administration to withdraw its approval of establishments, which are unable to apply the Hygiene Package in its totality. At the same time, they lament that the responsibilisation of producers by the Food Law does not mean the end of permanent inspection. When it comes to adopting the HACCP method, we can therefore see important differences between slaughterhouses [START_REF] Henson | Private sector management of food safety: public regulation and the role of private controls[END_REF]. The development of an HACCP plan is a good example of this confusion: in all of the establishments studied, the description of the veterinary service's activities was a real headache for the drafters. How to describe the inspection activity, which involves withdrawing carcasses unfit for consumption from the chain? Certain slaughterhouses considered that this made their industrial process safer, whereas others described this stage as a 'critical point' in as much as it could be performed in error. No general rule has been found to resolve this issue, which is now decided on a case-by-case basis.

More generally, the day-to-day operation of these establishments makes any strict task or responsibility sharing between inspectors and slaughterhouses impossible. To illustrate this point, let us take the case of one of the slaughterhouses in the study, which defined a CCP for the processing of soiled carcasses: they must be marked with an 'S' and withdrawn from the slaughter chain. But because it is too restrictive, this procedure is never applied, which leads to the veterinary team intervening:

In the cutting room, they have problems with salmonella and coliforms. There's too much faecal matter, too much negligence. I'm on the chain and for me it's obvious. They wanted to set up a system for faecal matter. The quality manager came: we had to remove them and no longer touch them. He finally gave up, because there were so many pigs being diverted off the chain that there was a rebellion by the workers: 'we've been working this way for 15 years, why change?' He gave in. So we went back to our own way of doing things: faecal matter removed to a greater or lesser extent, depending on the veterinary service. Faecal matter is the only critical issue in the HACCP. It's not normally up to us to deal with the problem, we're supposed to check that it's been properly dealt with. But the problem hasn't been dealt with. We're the ones dealing with the problem, more or less, through debridement. (Official auxiliary)

This example highlights the confusion between the acts performed by slaughterhouse workers and those carried out by the veterinary service: the latter cannot envisage limiting themselves to making observations without intervening when faced with clear cases of insalubrity. They therefore act as a substitute for the ineffective procedure and debride the carcasses where they are visibly soiled. In this way the carcasses are undoubtedly cleaner, but as slaughterers, inspectors are far from the roles and responsibilities defined for them by the ISO 17020 standard and the Hygiene Package.

The ecology of monitoring professions. In this new context, many veterinary inspectors feel that their occupation is being called into question. Indeed, the introduction of the HACCP logically supposes a food safety system which is constantly being improved, which is based on self-monitoring and which is validated from time to time by outside audits. This new factor is causing them to reconsider the need for permanent inspection, along with the types of skill and person required for the audits. Some of them believe that the certifying organisations might, or even should, replace them.

People expect us to carry out an inspection, based on comparison with a standard. Quality assurance tells us: 'You have a standard and you do your inspections in accordance with a standard, you do an audit and you say what is wrong compared to the standard. Then you add it all up and you make your conclusions'. Quite often on a certain number of points, things just aren't right! And you think, great! What's the point? WHAT'S THE POINT? Of saying: there's this point, that point and another point which don't conform. If that's all we do! What's the point? If it's an inventory so that the people know where they stand on their health barometer, what the hell is a PUBLIC inspector doing there? You privatise, end of story! (Official veterinarian)

There has been a significant increase in the number of slaughterhouse audits (with one every two weeks during the period of the study) be they appointed by the customers or auditors who come to grant or confirm a quality label (on the increasing number of audits, see [START_REF] Power | The audit society: rituals of verification[END_REF]. During the interviews, site directors all compared the different checks that are made, which lends weight to the notion of competition between inspectors from the veterinary service and outside auditors. One might therefore make the hypothesis of a jurisdictional conflict between the State inspectorate and audit organisations, e.g., between two professional groups who carry out the same tasks and whose aim is to maintain a settlement as food safety enforcers by the division of labour [START_REF] Abbott | The system of professions: an essay on the division of expert labor[END_REF]. To which aspects does this competition relate?

From the point of view of the slaughterhouses, we observed that the forms taken by the inspection were only rarely mentioned. The written exchanges are of course deemed to be clearer, but at the same time many of the interviews stressed the secondary nature of this evolution: 'It's intellectualism!' The comparison between different inspectors related more to the checklist and caused the slaughterers to judge the auditors as being more demanding than the veterinary inspectors.

Our main audits are for catering companies: Sodexo, Compass, Elior and Avenance if you prefer, along with Eurest. Second type of auditor, distributor's brands and slaughterhouses! And slaughterhouses! Because 15 per cent of our turnover comes from intermediate food products ... So that's a lot of people, and the biggest pains in the neck are the slaughterhouses. People like us in fact! Next to them, the veterinary service is peanuts! (Quality Manager, pig slaughterhouse)

We might nevertheless ask ourselves if this quality is proper to outside auditors or if it reflects the greater importance that slaughterhouses attach to the auditors appointed by their customers. The slaughterhouses highlight the firmer requirements for repairs to premises, revised circulation or more restrictive microbiological criteria. Veterinary inspectors recognise how these customer audits act as a spur to back costly investment decisions or to improve operating hygiene. Yet in his work on operators' attitudes during inspections, [START_REF] Muller | Visites à l'abattoir: la mise en scène du travail[END_REF] demonstrated that workers adapted to visitors in accordance with what they thought the latter's expectations to be: as far as they are concerned, it is not a case of the extent to which they must conform with regulations or specifications or with a checklist, but the extent to which they must adapt to the inspectors. When listening to what slaughterers say, one first and foremost remarks the priority that they set themselves: to adapt to the point of reference that they deem to be the most important -that of their customers.

Comparison between the different types of inspectors also depends on the quality of their specialist knowledge. All of the slaughterhouses visited stressed that they needed to see that the veterinarians possessed technical expertise, in order to monitor regulatory changes in small slaughterhouses or, for the highly industrialised abattoirs, to fall in line with a perspective of export and certification in relation to other countries (US, Japan, Korea, etc.). I'm in no way trying to say that the veterinary service in charge of the inspection and the slaughterhouses should be in connivance. That's not what I mean at all! They both have to play their role and take their responsibilities, and the veterinary service's power to inspect and then to punish if there are breaches, there's no question, we must have that and we must continue, because we can see that we don't do too badly when there are health crises. On the other hand, I don't think that should prevent us from having a closer relationship within the overall scheme of things. A stronger partnership and greater proximity would be good for economic development (we need to use the word without worrying about shocking people) and would therefore better valorise the French export. I think such proximity is really lacking. (Site Director, pig slaughterhouse) Finally, for the meat business operator, the point of comparing inspectors and auditors is not to determine who works the most efficiently, but to demand that the veterinary service do more than just inspect. Contrary to recent changes in the regulation and to what veterinary inspectors demand, they ask for a more comprehensive practice, more in line with the partnership between slaughterhouses and States described in certain developing countries [START_REF] Ponte | Bans, tests, and alchemy: food safety regulation and the Uganda fish export industry[END_REF]).

Conclusion

Our study offers possible explanations for this apparent paradox: that of proper application of the regulations but very poor results regarding operation and hygiene. Implementation of the Hygiene Package is not just a question of applying regulations and standards. It involves social relations in a world where inspectors and inspectees rub shoulders on a daily basis. But within the new regulatory framework, the definition of the roles of government officials and slaughterhouse operators is far from unambiguous. First of all, the inspectors continue to inspect every carcass in the chain, a domain where they have retained all of their prerogatives. For all other aspects, it is the slaughterhouse's HACCP plan which applies, a plan over which the veterinary inspectors hold little sway. Secondly, implementation of the Hygiene Package has changed the ways in which the veterinary service and slaughterhouses are organised and has led to a global movement which aims to clarify each party's responsibilities through formalisation. But on a day-to-day basis it is extremely difficult to always report on what everybody is doing and everyone thinks that it is illusory or even somewhat artificial to focus on recording systems. So, aware that such a strict and formal separation is futile, everyone is asking for better communication, contrary to the principles which drove the development of the food safety system. Finally, co-regulation describes the management of food safety by organising public and private actors around technical standards, which supposes the gradual removal of the sovereign figurehead of the State. On-site observation shows that recourse to the State occurs on a daily basis: in well-run abattoirs it remains the last resort in cases of clear danger; its presence prevents small slaughterhouses from being excluded, the latter providing a vital service to local farmers (organic farming, mountain breeders). We therefore find differentiated governance, which maintains the common objective of food safety for meat placed on the market, but which takes different forms.

The actors involved in the co-regulation are aware of this heterogeneousness, even if they do not necessarily appreciate its scope or modalities. Whilst everyone agrees that it needs to be reduced, they all have different solutions: as representatives of public authorities, veterinarians believe that it is through formalised legal instruments, of a similar nature throughout the country, that the harmonious application of regulations will be improved. Official auxiliaries believe their professional know-how when it comes to recognising wholesome meat and their professional conscience in promoting hygiene to be cornerstones for any homogeneous implementation of regulations. The largest slaughterhouses consider that health regulations should be based more on standards and that permanent slaughterhouse inspections are a source of distortion and should be gradually phased out. In this way, fair competition rules can be enforced throughout Europe, thus weeding out incompetent professionals whilst at the same time allowing the most competent to expand. Finally, the smaller slaughterhouses find it hard to see health regulations as anything other than a constraint on profitability. This conclusion is in turn an argument in favour of sociological work being carried out on these subjects in the different member states. critical limits for the potential hazards and safety measures, (4) Establish monitoring routines to ensure safety measures are working, (5) Establish appropriate responses if monitoring indicates a problem, (6) Establish accurate and detailed record-keeping system that documents problems and the remedial steps to be taken and (7) Establish a verification system that ensures the above steps are being followed. 4 As part of the prevention of BSE, tonsils have been removed from the food chain in Europe.

  2005 and MAP. Cartography Catherine Lefebvre (INRA-SAE2-MONA).

Figure 1 :

 1 Figure 1: Distribution of slaughter and numbers of abattoirs by French administrative regions

  a manufacturer of equipment for slaughterhouses. In this sentence, 'la Parmentière' refers to a paunch cleaning machine2 The founding text is Regulation 178/2002, or the 'Food Law', amended in 2004 by regulation 852/2004 on the general rules of hygiene for foodstuffs of animal origin; Regulation 853/ 2004 on the specific rules of hygiene for foodstuffs of animal origin; Regulation 854/2004 setting out the specific rules for the organisation of official inspections; Regulation 882/2004 on official inspections.

© 2013 The Authors. Sociologia Ruralis © 2013 European Society for Rural Sociology. Sociologia Ruralis, Vol 53, Number 4, October 2013

Although we have had a quality department for the last four years, now this suddenly arrives! We get the Hygiene Package, we get authorisations, so my staff and me, we are

The HACCP is a method based on seven principles: (1) Identify potential consumer health hazards, (2) Identify the control points where the identified hazards may occur, (3) Establish

Authority and power in the inspection process. Because it strengthens recognition of professional expertise, the Hygiene Package is also contributing towards veterinarians and official auxiliaries developing different conceptions of their occupation.

As they permanently share the life of workers in the slaughterhouse hall, the primary concern of official auxiliaries is to make their position as enforcers clear, as a condition for being able to exercise their functions:

In the same way, a rule enforcer is likely to believe that it is necessary for the people he deals with to respect him. If they do not, it will be very difficult to do his job; his feeling of security in his work will be lost. Therefore, a good deal of enforcement activity is devoted not to the actual enforcement of rules, but to coercing respect from the people the enforcer deals with. (Becker 1997, p. 158) They thus prefer to inspect from a position of authority, which takes concrete form in the immediacy of the observation and sanction, thus strengthening their position as enforcers. For example: an auxiliary notices a dirty or incorrectly labelled tub; he/she immediately asks an operator to clean it or change it. This type of action is only possible if the inspector and the person being inspected share the same reference point for the situation in question, such as a rule. With the Hygiene Package, it is the standards introduced by the slaughterhouse, brought together within the HACCP plan, which constitute the reference point for assessing situations and the slaughterhouse must correct the mistakes which are discovered in the implementation of its own procedures. According to the French ministry's directives, inspectors must 'ensure that the company has taken steps to properly verify the implementation of the HACCP plan. They must under no circumstances verify the plan itself in the company's place'. This new directive marginalises the capacity of inspectors to sanction errors in hygiene and changes the nature of their interventions. It makes it much harder to apply an immediate sanction.

The following extract illustrates the helplessness of an auxiliary who is confronted with a clearly irregular situation but who cannot see how to ensure that it is recognised as such. In accordance with practices prior to the Hygiene Package, he would like his word and his senses (sight, smell, touch) to be sufficient proof. But facts must now be proven in an entirely different manner, based on risk analysis:

The other day, with the pigs ... they'd made a chain which was much too fast. When the pig arrived for singeing, the flame worked for two seconds, and then, in fact, it became a continuous chain. The flame remained a flame, so the safety system went off and there was "glycolene" all over the pigs! The slaughterer finally said that we had to seize 22 of them. So I asked the veterinary surgeon: 'Why 22? Why not 25 or 28?' 'The slaughterhouse said so!'... They must have done some calculations I suppose ... but still! I said: we are the veterinary service and yet we're unable to say 'No, this one smells bad too! We'll take this one as well! ... That's why ... when it happens again and again ... We get discouraged. (Official auxiliary)

In this example, the auxiliary would like his authority to suffice for taking decisive and immediate action: you stop the chain, you withdraw the affected pig carcasses from consumption. As for veterinarians, they fall within the framework of the new Hygiene package and monitor the operator's risk analysis and procedures, which leads them to defer judgement and then to accept the conclusions of the slaughterhouse's assessment. In so doing, they come into conflict with their teams, who felt that an


Samira Khallouk

Roger Voisin

Cyril Van Ghelder

Gilbert Engler

Saïd Amiri

First, second, third, fourth Daniel Esmenjaud
email: daniel.esmenjaud@sophia.inra.fr

Histological Mechanisms of the Resistance Conferred by the Ma Gene Against Meloidogyne incognita in Prunus spp

The Ma gene from Myrobalan plum is a TNL gene that confers a highlevel resistance to all root-knot nematodes of major economic importance, including Meloidogyne incognita, M. javanica, M. arenaria, and M. enterolobii. The nematode behavior in the roots and the corresponding histological mechanisms of the Ma resistance to M. incognita in the resistant (R) accessions of the plum 'P.2175' and the interspecific hybrid P.2175 × almond-peach '35', carrying the Ma1 allele (Ma1/ma), were characterized in comparison with the susceptible (S) accessions in the plum 'P.2032' and the interspecific hybrid P.2175 × almond-peach '253' (ma/ma). Second-stage juveniles (J2s) were inoculated in micropropa-gated plantlets grown in soil substrate under controlled conditions at 25°C. Nematodes penetrated both R and S plants preferentially along the apical zone or close to the young lateral buds and moved via similar routes. Then they migrated into the cortex downward in the direction of the apex and turned up in the meristematic apical region to colonize the differentiating stele. In R accessions, motile J2s neither swelled nor developed into J3s, and initiation of feeding sites was never observed. This complete absence of gall symptoms is associated with cell necroses and corresponding hypersensitive-like reaction (HLR) phenotypes occurring either in the stele or in the meristematic apical region or in the cortex. Nematode attacks often disorganized the meristematic apical tissues of R accessions, which induced the development of subterminal lateral roots replacing primary terminal apices and, thus, provided an active resistance reaction to HLR damage.

Root-knot nematodes (RKNs) (Meloidogyne spp.) are polyphagous pests having a high impact on vegetable and fruit crops worldwide (1). These plant-parasitic nematodes are often the most important soil pests in Mediterranean countries under both field and protected cultivations [START_REF] Greco | Management strategies for nematode control in Europe[END_REF]. On a world scale, the most economically damaging RKNs are the Mediterranean and tropical species Meloidogyne incognita (Kofoid & White) Chitwood, M. javanica (Treub) Chitwood, and M. arenaria (Neal) Chitwood, which are highly polyphagous and reproduce through mitotic parthenogenesis [START_REF] Triantaphyllou | Cytogenetics, cytotaxonomy and phylogeny of root-knot nematodes[END_REF]. Those species are also major pests of fruit and nut production, particularly in the Prunus spp. crops such as peach, almond, and plum [START_REF] Nyczepir | Nematodes[END_REF]. Because chemical nematicides are prohibited in an increasing number of countries due to their side effects on the environment (2), the use of resistant cultivars is becoming a promising alternative for nematode control. Selection for RKN resistance in Prunus rootstock material has led to the characterization of several sources with more or less wide spectra [START_REF] Esmenjaud | Inter-and intraspecific resistance variability in Myrobalan plum, peach and peach-almond rootstocks using 22 root-knot nematode populations[END_REF][START_REF] Esmenjaud | Differential response to root-knot nematodes in Prunus species and correlative genetic implications[END_REF]. Among them, some Myrobalan plum clonal accessions (Prunus cerasifera Ehr; subgenus Prunophora) express a complete-spectrum, high and heat-stable resistance to RKN [START_REF] Esmenjaud | Effect of durable inoculum pressure and high temperature on root galling, nematode numbers and survival of Myrobalan plum genotypes (Prunus cerasifera Ehr.) highly resistant to Meloidogyne spp[END_REF]. In this plum, resistance to all tested RKN species and, notably, to M. arenaria, M. incognita, M. javanica, and M. floridensis (an American species reproducing on peach and almond sources), is conferred by the major and completely dominant Ma gene [START_REF] Esmenjaud | Inheritance of resistance to the root-knot nematode Meloidogyne arenaria in Myrobalan plum[END_REF][START_REF] Lecouls | Spectrum of the Ma genes for resistance to Meloidogyne spp. in Myrobalan plum[END_REF][START_REF] Rubio-Cabetas | Response of the Ma genes from Myrobalan plum to Meloidogyne hapla and M. mayaguensis[END_REF]. The positional cloning strategy for Ma has shown that it belongs to the Toll interleukin-1 receptor nucleotide-binding site leucine-rich repeat class of resistance genes [START_REF] Claverie | The Ma gene for complete-spectrum resistance to Meloidogyne spp. in Prunus is a TNL with a huge repeated C-terminal post LRR region[END_REF][START_REF] Esmenjaud | Genome mapping and molecular breeding in plum[END_REF].

Studies focused on the histological characterization of resistance to RKN have described diverse plant responses to nematode attacks ranging from strong early hypersensitive-like reactions (HLRs) leading to rapid juvenile death (Me3 in pepper) [START_REF] Pegard | Histological characterization of resistance to different root-knot nematode species related to phenolics accumulation in Capsicum annuum[END_REF] to late inhibitions of the formation of the giant cells associated with an incomplete development of the nematode (Me1 in pepper [START_REF] Pegard | Histological characterization of resistance to different root-knot nematode species related to phenolics accumulation in Capsicum annuum[END_REF] and Rk in cowpea [START_REF] Das | Histological characterization of root-knot nematode resistance in cowpea and its relation to reactive oxygen species modulation[END_REF]). In perennials, the few data obtained relate mainly to Prunus spp. carrying resistance to M. javanica in peach [START_REF] Malo | Nature of resistance of 'Okinawa' and 'Nemaguard' of the root-knot Nematode Meloidogyne javanica[END_REF] and almond-peach [START_REF] Marull | Resistance verification in Prunus selections to a mixture of 13 Meloidogyne isolates and resistance mechanisms of a peach-almond hybrid to Meloidogyne javanica[END_REF] and to coffee carrying the Mex1mediated resistance to M. exigua Goeldi (3).

In Myrobalan plum, a study on the dynamics of the penetration in resistant (R) and susceptible (S) accessions carrying Ma [START_REF] Voisin | Penetration, development and emigration of juveniles of the nematode Meloidogyne arenaria in Myrobalan plum (Prunus cerasifera) clones bearing the Ma resistance genes[END_REF] has been conducted. However, in this case as in other annuals and perennials, no precise histological documentation of nematode behavior in the plant during compatible and incompatible reactions is known. Here, we report a study of a detailed histological characterization of the nematode routes and of the histological mechanisms of resistance conferred by the Ma gene from Myrobalan plum using R (P.2175) and S (P.2032) Myrobalan plum clonal accessions. In order to validate our results in a different genetic background, an equivalent study was also conducted using Ma-resistant and ma-susceptible interspecific hybrids between P.2175 and almond-peach.

MATERIALS AND METHODS

Plant material. Four clonal accessions were used in this study: P.2175 and 35 (carrying Ma) and P.2032 and 253 (lacking Ma). Accessions P.2175 and P.2032 are pure natural Myrobalan plum cultivars originating from Romania and southeast France, re-spectively, whereas accessions 253 and 35 are interspecific hybrids between P.2175 and almond-peach (Garfi × Nemared) [START_REF] Li | The Rop GTPase switch controls multiple developmental processes in Arabidopsis[END_REF] and (Garfi × Nemared) [START_REF] Esmenjaud | Differential response to root-knot nematodes in Prunus species and correlative genetic implications[END_REF] , respectively (Table 1). P.2175 was selected as rootstock for its high RKN resistance conferred by the Ma gene to the most economically damaging species (M. arenaria, M. incognita, M. javanica, and M. floridensis) [START_REF] Cetintas | Pathogenicity and reproductive potential of Meloidogyne mayaguensis and M. floridensis compared with three common Meloidogyne spp[END_REF][START_REF] Lecouls | Spectrum of the Ma genes for resistance to Meloidogyne spp. in Myrobalan plum[END_REF] and for its favorable agronomic features such as efficient vegetative propagation and graft compatibility with many fruit cultivars of peach.

Prunus plantlets obtained from in vitro, 8-cm high, were acclimatized in perlite trays for 2 weeks and repotted into 50-ml plastic tubes containing a mix of siliceous sand, sterile silt-clay soil, and peat moss (4:3:1, vol/vol/vol) and maintained in the growth chamber at the ambient temperature of 25°C with a photoperiod of 16 h.

Animal material. Histological studies were conducted on plant material inoculated with the isolate M. incognita 'Morelos' (Mexico). This nematode isolate had been reared from a singlefemale egg mass. For the production of the inoculum, secondstage juveniles (J2s), <72 h old, were collected from egg masses recovered from roots of susceptible tomato 'St. Pierre' previously grown in greenhouse.

Histological experiments. Inoculation was performed around the plant collar into four holes (1 ml of water per hole), 2 cm deep. Plants of the R accessions (P.2175 and 35) were inoculated with a water suspension containing 20,000 J2s. Indeed, in a previous study [START_REF] Voisin | Penetration, development and emigration of juveniles of the nematode Meloidogyne arenaria in Myrobalan plum (Prunus cerasifera) clones bearing the Ma resistance genes[END_REF] with equivalent plant characteristics (same age, also obtained from in vitro), mean number recovered in the total root system of plants of P.2175 inoculated with 2,500 J2s was ≈200 J2s per plant (for 400 estimated total apices per plant). This number of nematodes per apex was critically low for an accurate description of nematode behavior inside the roots of resistant plants; therefore, the level of 20,000 J2s per plant was chosen to allow the penetration of higher numbers of J2s. Plants of S accessions (P.2032 and 253) were inoculated with 5,000 J2s. This inoculum level was chosen on the basis of a previous methodological study [START_REF] Esmenjaud | Assessment of a method using plantlets grown previously from in vitro for studying resistance of Prunus cerasifera Ehr. (Myrobalan plum) to Meloidogyne spp[END_REF] on the accession P.2032, with equivalent plant characteristics, showing a limited intraspecific competition between nematodes in these conditions.

Terminal root fragments, 10 mm long, were collected 1, 2, 3, 5, 8, 12, 17, 23, 30, and 40 days postinoculation (dpi). After collection, root tips were washed with tap water and observed under a binocular microscope for nematode penetration and root galling. Then, infected roots were fixed overnight at 4°C in 2% glutaraldehyde, rinsed with 'Pipes' (Sigma-Aldrich), and dehydrated by passing through a graded ethanol series at 1, 5, 30, and 50% (2 h each); 70% (overnight); and 90% (2 h) followed by 100% in three successive independent immersions of 2, 1, and 1 h. Infiltration and embedding were done with epoxy resin (Teknovit 7100 kit; Kulzer Friedrichsdorf, Germany). Sections, 5 µm thick, were cut using a microtome (Adamas Instrument en BV, The Netherlands). Successive serial sections were shared in two equivalent groups and mounted on poly-L-lysin slides. One group of slides was stained for 30 s with toluidine blue in 0.1 M sodium phosphate buffer (pH 5.5), rinsed with ultrapure water, and dried. Stained reactions were examined under a Zeiss Axioplan light microscope equipped with a Nikon Cooplix 995 digital camera. In order to reveal aromatic compounds, the other group of slides was kept unstained and examined using the microscope with a UV filter set at 365-nm excitation and 420-nm emission lengths. Yellow and orange autofluorescence revealed the presence of phenolic compounds and a pale blue autofluorescence revealed the presence of lignified cells.

Total numbers of root fragments observed per date ranged from 6.8 (accession P.2175) to 8.7 (accession 253), totaling ≈3,000 slides when all observation dates were considered for the four accessions.

Macroscopic reaction to nematode attacks in resistant accessions. To describe the resistance reaction to nematode attacks at the macroscopic level, accession 35 was monitored at 17 dpi after inoculation (20,000 J2s) in comparison with uninoculated controls. Five plants of each modality were removed from the tube and their entire root system was carefully washed free of substrate. Among the entire root system, all single terminal fragments or tips (i.e., from the apex up to the junction of the fragment with another root or rootlet) were cut, recovered, and selected for further examination. In this examination, fragments with terminal buds emerged <2 mm from their root extremity were considered as a single fragment or tip. In inoculated and uninoculated modalities, all fragments selected as described above were examined for their proportion of tips that harbored such lateral buds (i.e., emerged <2 mm from the extremity) and their number of terminal apices per fragment. From these data, the percentage of tips with lateral buds and the mean number of buds per root tip were compared between both modalities. The same procedure was repeated at 35 dpi in order to compare the evolution of the phenomenon.

RESULTS

Observations reported hereafter deal successively with root penetration, migration, and plant reaction to nematodes attacks. In each case, they are described first on Myrobalan plum (i.e., in both the resistant P.2175 and the susceptible P.2032 accessions) and then on the Myrobalan × almond-peach hybrids (i.e., in both the resistant 35 and the susceptible 253 accessions).

Root penetration. J2s were observed as penetrating the roots of P.2175 from 1 dpi, with a preferential location close to the meristematic region of primary root apices and a secondary location next to lateral buds (Fig. 1A andB). Penetration into the elongation zone, upward of the apical region, was less frequent. In the S accession P.2032, penetration occurred in similar locations (Fig. 1C andD). In both accessions, penetrating J2s, although decreasing in numbers, were visualized up to 17 dpi. In the R or S interspecific accessions, locations of penetration were equivalent to pure Myrobalan plum (data not shown). Several nematodes grouped under the first cell layers were observed, illustrating that penetration often occurred in only a few points of the root surface (Fig. 1E andF).

Migration. In the roots of the resistant accession P.2175, the J2s that had just gone through the epidermis and reached the cortical region always oriented themselves downward, in the direction of the root tip (Fig. 1G andH). We observed that this longitudinal migration to the apical meristematic cells could occur more or less deeply in the cortical tissue but was always limited by the endodermis (Fig. 1H andP). Although J2s were shown to migrate intercellularly, needing a channel for this corresponding approximately to their diameter, they occasionally created big spaces between cell layers. This was particularly marked when the route opened by a first juvenile was used by several others (a frequent observation) that were located either in a single file or more crowded all together (Fig. 1J andK). When J2s reached the meristematic apical region of the roots, they turned around in the central part of the apex (Fig. 1H, L, andN, with details of J2s rotation in M and O) and migrated upward between the cells toward the differentiated vascular cylinder (Fig. 1I andQ). The first J2s reaching the vascular cylinder were observed at 3 dpi. However, a complete migration of J2s up to the differentiated region of the stele was not always observed. In many cases, nematodes remained in the meristematic terminal region and were surrounded by a disrupted structure of this root zone (Fig. 1P andQ). In the roots of the susceptible accessions P.2032 and 253, the early migration route of the motile J2s appeared equivalent to that of the resistant accessions P.2175 and 35, with a downward migration into the cortex, a rotation in the meristematic apical region, and an upward colonization of the vascular cylinder. Nevertheless, in S accessions, destruction of the tissues was never observed around moving nematodes.

Compatible interaction in susceptible accessions. In P.2032, external macroscopic observations of roots revealed the first galls at 5 dpi (Fig. 2A). At that date, observations of gall sections showed swollen juveniles in the vascular cylinder which were already inducing feeding sites around their head. However, there still existed some J2s which had not yet reached the vascular cylinder. At 8 dpi, the galls were large and well developed as a result of the hypertrophy of the giant cells and the hyperplasia of surrounding cells. At 17 dpi, all of the developmental stages of the nematodes (J2, swollen J2, J3, J4, and female) were simultaneously present in the galls (Fig. 2C to F). The first female with an attached egg sac was observed at 23 dpi (Fig. 2F andG). Eggs were visible on the root surface in the protective, gelatinous matrix (Fig. 2H). At 40 dpi, the presence of the filiform males was detected in the tissues (Fig. 2I).

HLR in resistant accessions. Examination of ≈920 root sections of the accessions P.2175 and 35 showed that all J2s recovered inside the roots remained filiform (body diameter of 12 to 15 µm) and that there was no formation of giant cells. Microscopic observations of stained root sections revealed darkblue color characteristic of an HLR around the nematode at different dates. The earliest HLR was characterized at 3 dpi in our study (Fig. 3A andB). Such reactions occurred in either the vascular cylinder (Fig. 3A andG), the meristematic or bud regions (Fig. 3C andE), or the cortex (Fig. 3I). The intensity of staining seemed to be higher at later dates (40 dpi) (Fig. 3I andJ). When root sections were examined under UV light, the yellow or orange autofluorescence (Fig. 3D, F, andK), exhibiting the presence of aromatic compounds, was seen within the cells and in cell walls. In our figures, we could observe clear signs of nematode death at 23 dpi (Fig. 3G andH). Initiation of lateral buds and development of replacement lateral roots at the apices of resistant accessions under nematode attacks. In Ma-resistant accessions, observations of development of roots in apical regions consecutive to nematode attacks showed the emergence of newly formed lateral roots from buds formed just upward of the apices (Fig. 3L to P). These subterminal lateral roots emerged from the stele as a consequence of disorganization of tissues observed in the meristematic terminal regions (Fig. 3Q to S). Newly formed lateral roots were either single or double (symmetrical). When the root was single, it took the same growth direction as the damaged root and appeared to be replacing it. Numerical evaluation of root development showed that the proportion of tips with lateral buds is highly significantly different (more than twice) in the inoculated plants in comparison with the negative controls (χ 2 = 75.2; P < 0.001). The number of lateral buds per root tip is also highly significantly different, being more than doubled in the presence of the nematodes (χ 2 = 49.4; P < 0.001] (Table 2). Moreover, values of neoformation of roots are equivalent after 17 and 35 days (χ 2 = 0.26 and 0.28, respectively, for proportion of tips with lateral buds and number of buds per root tip) (Table 2).

DISCUSSION

The first aim of this study was to compare RKN penetration and subsequent development in Ma-resistant and ma-susceptible accessions of Prunus spp. In the four tested accessions, we observed J2s initiating their penetration <48 h after inoculation into the soil. Although their numbers decreased, penetrating nematodes were observed up to 17 dpi, stating that motile J2s can survive in the substrate for more than 2 weeks. Our results, obtained in both Myrobalan plum and interspecific genetic backgrounds, are in agreement with previous data from Voisin et al. [START_REF] Voisin | Penetration, development and emigration of juveniles of the nematode Meloidogyne arenaria in Myrobalan plum (Prunus cerasifera) clones bearing the Ma resistance genes[END_REF], in which equivalent numbers of M. arenaria were recovered in the roots of the susceptible P.2032 and resistant P.2175 accessions at 1 and 2 dpi. Consequently, we confirm here that the Ma gene does not induce an early resistance phenomenon due to a physical or chemical barrier to penetration [START_REF] Cook | Resistance and tolerance[END_REF][START_REF] Huang | Formation, anatomy and physiology of giant cells induced by root-knot nematodes[END_REF]. This situation is contrary to CM334 pepper (carrying the Me7 gene), in which J2s are preferably blocked in the epidermal layer of cells [START_REF] Pegard | Histological characterization of resistance to different root-knot nematode species related to phenolics accumulation in Capsicum annuum[END_REF] and might trigger an active early post-penetration biochemical defense mechanism.

We have shown that, in material carrying Ma as well as in material lacking it, penetration occurred preferentially in the apical region or in the vicinity of the lateral buds (i.e., in areas where cells are the youngest and are expected to facilitate nematode mobility). Moreover, penetration sites were shown to be often shared by several juveniles which afterwards used the same routes inside the plant. After penetration, J2s migrated intercellularly, separating cells at the middle lamella in the cortical tissue. This intercellular migration of Meloidogyne spp. described in many plant species [START_REF] Endo | Ultrastructural investigation of clover roots during early stages of infection by the root knot nematode Meloidogyne incognita[END_REF][START_REF] Trudgill | Apomictic, polyphagous root-knot nematodes: Exceptionally successful and damaging biotrophic root pathogens[END_REF] is confirmed here. This process appears to include both mechanical forces and enzymatic secretions from the nematodes [START_REF] Williamson | Nematode pathogenesis and resistance in plants[END_REF]. In both R and S accessions, we have observed that, once nematodes had penetrated the terminal zone, they never moved transversally in the roots through the endodermis and the pericycle (to directly reach the protoxylem and protophloem cells where they should become sedentary and fix to initiate their feeding site) [START_REF] Huang | Formation, anatomy and physiology of giant cells induced by root-knot nematodes[END_REF]. Our hypothesis is that, because of the presence of specific structures called the casparian strip reinforcing the radial and transversal walls of endodermal cells [START_REF] Esau | The root[END_REF], J2s are unable to dissolve the middle lamella between these cells for direct access to the vascular cylinder. Thus, they might be obliged to migrate first downward in the direction of the meristematic region, turn in the opposite direction, and, last, to move upward in the direction of the vascular cylinder. Even though the same route has been described in the compatible reaction between M. incognita and Arabidopsis thaliana using immunolabeling techniques at the cellular level [START_REF] Gravato-Nobre | Meloidogyne incognita surface antigen epitopes in infected Arabidopsis roots[END_REF], we have shown here the first example of this nematode behavior in an accession carrying a resistance gene.

In resistant clones (P.2175 and 35), the juveniles neither swelled nor developed but remained filiform. This shows that the Ma gene completely prevented the fixation of the motile J2s in cells from the different plant tissues through which they move and, particularly, from the vascular cylinder, thus depriving it of nutrients and inhibiting further development into inflated stages. Thus, in this plum gene, we observed a situation different from Malo [START_REF] Malo | Nature of resistance of 'Okinawa' and 'Nemaguard' of the root-knot Nematode Meloidogyne javanica[END_REF] with M. javanica, who reported the presence of thirdstage juveniles inducing incomplete giant cells at 4 dpi in the resistant peach rootstocks 'Okinawa' and 'Nemaguard'. Identical responses of partial nematode development and incomplete giant cell formation were also described by Marull et al. [START_REF] Marull | Resistance verification in Prunus selections to a mixture of 13 Meloidogyne isolates and resistance mechanisms of a peach-almond hybrid to Meloidogyne javanica[END_REF] in interspecific Prunus material derived from Nemaguard. The histological study on the Mex-1 gene conferring resistance in coffee (Coffea arabica) to M. exigua also showed that J2s swelled to J3 to J4 and initiated their feeding sites at 10 to 14 dpi (3).

Within roots of the susceptible P.2032 and 253 accessions, J2s induced giant cells among the vascular cylinder. As classically described, the giant cells contained highly condensed cytoplasm and enlarged nuclei (with marked nucleoli) resulting from mitosis uncoupled from cytokinesis [START_REF] Davis | Parasitism proteins in nematode-plant interactions[END_REF][START_REF] Huang | Formation, anatomy and physiology of giant cells induced by root-knot nematodes[END_REF]. Concurrent swelling and division of cells around the head of the nematode led to the formation of galls and the distorted root structure characteristic of Meloidogyne spp. infection. Our data at 25°C established that, from the motile J2 stage (inoculation date) and the development of the nematode up to the detection of the first female lasted <17 days, and up to the first female carrying an egg mass lasted <23 days. Egg masses, released on the root surface in a protective gelatinous matrix, were less apparent than in tomato, presumably because of the rigidity of tissues due to the synthesis of lignin.

The present histological study clearly showed that resistance conferred by the Ma gene is associated with cell necroses (arresting the nematode development) and the corresponding HLR phenotypes occurring in either the vascular cylinder, the meristematic apical or bud regions, or the cortex. Differential staining with toluidine blue revealed dark-blue and blue-green coloration of HLR cell compounds and suggested accumulation of phenolics [START_REF] Locquin | Manuel de Microscopie[END_REF]. Under UV light, some compounds emitted yellow-orange fluorescence with a lower intensity than in resistant pepper CM334 [START_REF] Pegard | Histological characterization of resistance to different root-knot nematode species related to phenolics accumulation in Capsicum annuum[END_REF], in which a high content of phenolics such as chlorogenic acid has been detected. In resistant accessions P.2175 and 35, the first signs of cellular necrosis were localized in the vascular cylinder at 3 dpi. Further investigations are required in order to understand more accurately the underlying mechanisms involved in the HLR mediated by the Ma gene in Prunus spp. at both cellular and molecular levels. Another interesting feature identified in resistant material was the emergence of new lateral roots near the infected root tips. Malo (24) also reported the same feature on resistant cultivars of peach. We could show that each of the criteria "percentage of tips with lateral buds" and "mean number of buds per root tip" were increased in infected plants. This rapid induction of lateral roots consecutive to the nematode attack, by contributing to the replacement of damaged apices, is involved in the overall resistance of the plant against the RKNs. Moreover, values of these criteria were equivalent after 17 and 35 days, which suggests that plant response to nematode effect reaches a maximum before or at 17 days. This is in agreement with our results that showed that J2s penetrated up to this date, although in decreasing numbers, and, consequently, their destructive activity is expected to be lower after it.

In Arabidopsis, this ability to create new lateral roots has been shown to require a repolarization of auxin (IAA) by carriers termed PIN proteins [START_REF] Jallais | The retromer protein VPS29 links Cell polarity and organ initiation in plants[END_REF]. These carriers cycle between the basal and lateral sides of the cell: this polarity change and subsequent formation of new axes of development consecutive to root cell division is under the control of the protein VPS29 from the "retromer" complex. An overexpression of the ROP2 signal transmitter in cells accumulating auxins [START_REF] Li | The Rop GTPase switch controls multiple developmental processes in Arabidopsis[END_REF] might also be linked to the formation of lateral meristems. In Prunus spp., homologous genes of these proteins presumably exist and the characterization of their expression in the root tissues carrying the Ma gene would provide evidence of their involvement in the lateral root formation consecutive to nematode attacks.

Fig. 1 .

 1 Fig. 1. Itinerary of second-stage juveniles (J2s) in the resistant (P.2175 and 35) and susceptible (P.2032) accessions. A and C, Apical penetration. B and D, Penetration next to lateral buds. E and F, Penetration in the same points either in the apical area (arrow head) or in the bud, respectively. G to I, Different steps of the migration at 3 days postinoculation (dpi). G, J2 having migrated in the cortex. H, J2 channel in the inner cortex limited by the endodermis (arrow); J2 leaving the cortex and penetrating the meristematic apical region (arrow head) before turning upward in the direction of the vascular cylinder. I, J2 in the vascular cylinder (the arrow points the nematode head). J, Two J2s migrating intercellularly along the same route (8 dpi). K, Magnification of J. L and N, J2 moving from the cortex into the apical meristematic region (17 dpi). M and O, Magnification of L and N, respectively. P and Q, Several J2s causing disruption of tissues in both P, the cortical and meristematic region and Q, the meristematic region (arrows) at 8 dpi. Arrowheads point to the longitudinal channels caused by nematodes during migration in the P, cortex and Q, vascular cylinder; ep = epidermis, co = cortex, vc = vascular cylinder, ne = nematode, mb = median bulb, st = stylet, bd = bud. Bars = 100 µm.

Fig. 2 .

 2 Fig. 2. Compatible interaction in susceptible accessions (P.2032 and 253). A, Galls on apices at 5 days postinoculation (dpi). B, Swollen second-stage juvenile (J2) in the stele at 3 dpi. C to F, Sections of galls showing feeding sites with different developmental stages of the nematodes at 17 dpi: C, J3; D, J4; E, young female; and F, female with attached egg mass. G, Posterior end of female (arrow) emerged at the gall surface at 23 dpi. H, Gelatinous matrix containing eggs (separated from the egg mass) at 23 dpi. I, Feeding site with vacuolated giant cells and females at 40 dpi. Note filiform male still coiled within the J4 cuticle; ep = epidermis, co = cortex, vc = vascular cylinder, ne = nematode, mb = median bulb, st = stylet, fe = female, mt = matrix, eg = egg, m = male, * = giant cell. Sections were stained with toluidine blue. Bars = 100 µm (except A, G and H, where bars = 500 µm).

Fig. 3 .

 3 Fig. 3. Incompatible interaction in resistant accessions (P.2175 and 35). A, C, E, G, and I, Sections stained with toluidine blue showing hypersensitive-like reactions (HLRs) associated with second-stage juveniles (J2s). B, H, and J, Magnification of A, G, and I, respectively. D, F, and K, Unstained sections observed under UV showing yellow and orange fluorescence illustrating HLR around J2s. L to P, Lateral roots neoformed (arrowheads) near damaged apices (arrows). Q, R, and S, Sections showing damaged meristematic apical tissues (arrow) and correlative emergence of lateral roots (section S is unstained and observed under UV); ep = epidermis, co = cortex, vc = vascular cylinder, ne = nematode, mb = median bulb, st = stylet. Bars = 100 µm (except L to P, where bars = 500 µm).

TABLE 1 .

 1 Plant material used in the study

	Gene	Myrobalan plum	F1 interspecific material
	Resistant (Ma1/ma)	P.2175

[P.2175 × (Garfi × Nemared) 15 ] 35 Susceptible (ma/ma) P.2032 [P.2175 × (Garfi × Nemared) 22 ] 253

TABLE 2 .

 2 Effect of nematode attacks on lateral root bud emergence in the resistant accession 35

	Inoculated plants a

a Abbreviation: dpi = days postinoculation. b Percent = number of apices harboring lateral buds emerging less than 2 mm from the extremity/total number of apices. c χ 2 = 75.2 (P < 0.001). d χ 2 = 0.26. e Total number of lateral buds (as considered above)/total number of apices. f χ 2 = 49.4 (P < 0.001). g χ 2 = 0.28.