Lourdes Monterrubio Ibáñez

The Interstice Between Reality

La Morte Rouge (soliloquio) by Víctor Erice. From Trauma to Fraternity. The Interstice between Reality and Fiction

Introduction

After the premiere of El sur (1984), Víctor Erice declared about his next projects: 'I have often been tempted to drift towards a fragmentary cinematic structuring, the intimate diary, the essay, the reflection, perhaps with a touch of fiction' (Molina Foix, 1984: 51). Two decades later, in the opening of the exhibition Correspondences Erice -Kiarostami, the filmmaker then used the concept 'fraternity between reality and fiction' 1 to describe his way of understanding the cinematic experience. While Dream of Light/El sol del membrillo (1992) is a precursor of/to a new contemporary cinema based on this fraternity (Monterrubio Ibáñez, 2018b), the essayistic reflection becomes a filmic form in this correspondence between the filmmakers and, very especially/in particular, in La Morte Rouge (soliloquio) (2006). The temptation Erice confessed becomes an attempt, generating the two polarities theorised by Alain Ménil in relation to the essay film, temptation-attempt and objectivity-subjectivity:

There is no essay which is not, in some way, the experience of its own adventure, which is not at the same time a search, an investigation or an inquiry apropos of or on the occasion of, the occasion of an invention, invention of its own method and its own path (Ménil 2004: 101, italics in the original).

The work responds to the most demanding definition of this filmic form, which would materialise/embody the thinking process and the self-reflection of a subjectivity or 1 Expression used by Víctor Erice in an interview in El Cultural, 15th May 2002: http://www.elcultural.com/revista/cine/Victor-Erice/4789 Concept also presented by the filmmaker at the round table presentation of the exhibition Erice -Kiarostami. Correspondence at La casa Encendida, Madrid 5th July 2006.

subjectivities through the hybridisation of fiction, non-fiction and experimental cinema.

Therefore, the 'presence of the author's self, of the essayist' and 'the revelation of a thinking in act' (Moure, 2004: 37) are necessary conditions that determine the enunciation of the film. Thinking in act also defined by Josep Maria Català: 'a reflection through images, carried out through a series of rhetorical tools that are constructed at the same time as the reflection process ' (2005a: 133), in which 'the author is always linked to this process [of thinking] and it is not possible to flee from that presence, since the work itself is nothing other than the verification of it' (Català, 2014: 143).

Enunciative device: the soliloquy

Erice's work is inscribed in the continuation of the film essay/essay film practices of cinematic modernity. The voice-off -following Michel Chion's nomenclature and definition-of the essayist (or of his/her interpreter, as in the case of Chris Marker) generates the filmic enunciation by means of clearly coded devices that I call intermedial forms (the letter, the diary, the self-portrait, etc.), already present in their titles on many occasions. We find an obvious verification of this phenomenon in French cinema, of which I present some relevant examples:

-The letter2 :

-The diary:

-The (self)-portrait:

-The scenario/script: Regarding the devices, Erice chooses the soliloquy, defined in Spanish as: 'interior or aloud reflection made alone', to turn it into a 'tool for investigation in the filmmaker's own cinematic memory' (Balló, 2012: 14). And not only in the cinematic one, but also in childhood memory and historical memory, allowing the filmmaker 'to fuse, in a single gesture, the most intimate and personal memory with the density and weight of history' (Zunzunegui, 2014: 57). The soliloquy is the perfect device to create a 'memory operations laboratory' (Berthet, 2011: 86), understanding the latter as a 'practice and a semiotic-discursive process', 'a dynamic cognitive process that involves the symbolic construction of meaning, the semiosis' (Ricaurte, 2014: 48, 53). In this way, the articulation of creative, emotional, historical and collective memory makes filmic thinking possible: 'Conception, therefore, of memory [...] as something alive and organic that is reached from the present and reconfigured from it [...] renouncing the document to access to experience and truth, to the aesthetic gaze on one's own life' (González, 2006: 200).

The soliloquy allows the author to generate a parataxic thinking (Català, 2014: 206) that also responds to the notion of parataxic syntax exposed by Jacques Rancière (2009: 48) and to the concept of sentence-image in relation to its audiovisual essence: 'The sentence is not the sayable and the image is not the visible. By sentence-image I intend the combination of two functions that are to be defined aesthetically -that is, by the way in which they undo the representative relationship between text and image" (46). This sentence-image will transit between the dialectical and symbolic poles: 'between the clash that a doubling of measurement systems operates and the analogy that shapes the great community, between the image that separates and the phrase that tends towards phrasing continuous' (58). Both procedures can intermingle and 'verge on the indiscernible' (60). The author's creative memory then travels to his childhood to reflect on his first cinematic experience, the screening of the film The Scarlet Claw (Neill, 1944) present images, in colour video, with which the film begins and ends; -B/W archive images of the time, photographic and filmic;

the filmmaker's personal photographs;

the fictional recreation of the childhood experience in B/W; images from The Scarlet Claw, still and in motion.

Erice's essayistic experience thus materialises/embodies the montage of propositions:

'to look at the image again, to denature its original function (narrative, observational) and to see it as representation, not to only read what it represents' (Weinrichter, 2007: 28). As Català indicates with respect to the complex image, it 'breaks the mimetic link that the image traditionally maintained with reality and replaces it with a hermeneutical link: instead of an epistemology of reflection [mimesis], an epistemology of inquiry is proposed ' (2005b: 642). The filmmaker's voice, present together with the different visual materials and soundsincluding Arvo Pärt's music and the piano composition, Música callada, by Federico Mompou-, organises and goes through the different materials, causing their transformation and allowing the revelation. The filmmaker's vocal performance also traverses different mental spaces: the adulthood essayistic reflection, the poetic sphere of intimate memory and the emotional territory of childhood experience.

Interstitial thinking

Returning to Gilles Deleuze's postulates (1985) on the interstice, and its condition as a germ of cinematic thinking (Monterrubio Ibáñez, 2018a), and coinciding with Laura Rascaroli's proposal: 'the essay film, as a thinking cinema, thinks interstitially [...] to understand how the essay film works, we must look at how it forges gaps, how it creates disjunction ' (2017: 11). The cinematic thinking of La Morte Rouge is generated from four disjunctions. The first, of a temporary nature, faces the present of 2006 with the past of 1946. The second, of an identity character, fractures the self between the present adult identity in the first person and the past one, placing the five-year-old boy in a third person which objectivizes him, subjecting the intimate autobiography to analytical scrutiny and turning it into a story too, into fictional reconstruction of childhood experiences. The third, the disjunction between the reality of the boy and the cinematic fiction, paradoxically becomes indiscernible for the childhood experience, causing the trauma that can be reversed only through the artistic experience. Finally, a last disjunction between fiction and essay materialises, between the absent work and the present one, between the fiction of The Spirit of the Beehive/El espíritu de la colmena (1973) and the reflection of La Morte Rouge, configuring both works as a diptych. As Jean-Louis Leutrat (2004) indicated, this diptych structure is an ideal mechanism for filmic thinking, as confirmed by the Godardian experience in some of the previously cited titles (Monterrubio Ibáñez, 2017). The film is constructed through the articulation of these four disjunctions in the three reflection levels already mentioned: intimacy, cinema and history. I will analyse below the audiovisual procedures, the rhetorical elements, that generate this 'interstitial thinking' (Rascaroli, 2017: 190).

From trauma to fraternity

The introduction of the soliloquy presents us its theme and the rhetorical elements through which the film will develop: the cross-fade, the panoramic movement within the still image and the fade to/from black. The temporal disjunction is transited/traversed through a cross-fade chained in its classic functionality, which informs the passage of time in which the transformation of the Gran Kursaal Casino into a cinema takes place. The panoramic movement, for its part, introduces the research, the search that allows the generation of hypotheses. Finally, the fade to/from black materialises/embodies reflective pauses around the different addressed interstices.

The reality of the Gran Kursaal cinema slides towards the fiction that will emerge from within, that of the screening of The Scarlet Claw, through a first symbolic sentenceimage: that of the spinning roulette wheel. This is the only fictional image about the Kursaal's past and it will take full meaning a posteriori, materialising/embodying an equally defining décalage of the essay film thinking in act. Erice turns the dialectics between fiction and reality, through the semiotic-discursive process of memory, into a circular structure, without beginning or end, which has its perfect metaphor in the casino roulette. Its rotating movement causes its image to dissolve, just as the cross-fade interweaves the different filmic materials, as materialisation of the memory process.

Thus 'a territory in which history and imagination incessantly settle their accounts in permanent dialogue' (Zunzunegui, 2014: 57) arises. The initial dialogue, the various resonances that emerge among the different materials, becomes, during reflection, the fusion of fiction and reality that occurs in childhood perception. The disappearance of the interstice between fiction and reality, the indiscernibility between both spaces, provokes the childhood trauma around the cinematic experience, about which Alain Philippon reflected: "between the child and cinema something happens that is of the order of trauma, with all the more reason -although not necessarily-when the images in question are images of fear" (1999: 17). Erice himself writes after the death of the French critic:

For him, trauma, always associated with the early, childhood viewing of a film, was the place of origin, the one from which history starts to deploy its fiction. Not only the place from which one comes, but also the destination towards which, without knowing it, one is heading. (Erice, 1999: 16) The childhood trauma, therefore, will deploy its fiction in The Spirit of the Beehive and will also place/fix its destination in the essay film La Morte Rouge, 'revealing the biographical keys hidden within The Spirit of the Beehive (1973), acquiring the air of intimate confession around the space of origins' (Quintana, 2008: 100). This diptych structure is thus configured as an interpretive key to Erice's film career.

After the second fade to black, the boy's portrait appears. The trauma object of the soliloquy, which is explained next, is enunciated around a third grammatical person that distances the identification with the essayist: 'At the time that boy was five years old.

For a long time, I avoided putting a date to that crucial experience, which thus remained in an undefined spot in the lawless territory of earliest childhood'. This identity disjunction is accompanied, however, by the identification between this real boy character and his fictional incarnation, the little Ana from The Spirit of the Beehive. In the film, Ana appears for the first time, also accompanied by her sister, during the cinematic experience of Frankenstein (James Whale, 1931). Faced with the trauma caused by the indiscernibility between fiction and reality, the absence of interstice, the filmic creation of a fiction seeks to manage it.

Then, the presentation of The Scarlet Claw is accompanied by still images that are associated with the film and that, however, on different occasions, belong to other films of the series: The Adventures of Sherlock Holmes (Alfred Werker, 1939), The Pearl of Death (Roy William Neill, 1944). In this way, the symbol-image of fictional fear, which will be repeated at key moments of the reflection, turns out to be a still from Dressed to Kill (Roy William Neill, 1946): 'fragment of a broken mirror from a moment of our life, a fragment of the puzzle of our Imaginary" (Philippon, 2002: 18-19). As it will happen on different occasions, the realist conception that materialises in the fiction film disappears in the filmic essay, in which the work of creative memory, its semioticdiscursive process, and the research work of the complex image, abandons the realist paradigm in favour of that epistemology of inquiry.

At this moment, the cross-fade becomes the first rhetorical element of this process of indiscernibility. It materialises/embodies the shift of fear from fiction to historical reality, that of the Spanish postwar period and the Second World War: 'for the public The Scarlet Claw was mainly just one more scary film. Except that, in this instance scariness spread forth beyond the screen, prolonging its echo in the ambience of a devastated society.' Again, the documentary image of the essay film evokes and invokes the fictional creation of The Spirit of the Beehive. What for the adult individual is a leisure activity through which to manage the fear imposed by reality, for the boy, still unaware of the differentiation between fiction and reality, becomes trauma. The cross-fade used between the previous frames is now slowed down to cause the superimposition, and to produce a symbolic sentence-image of it: superimposition between the horror of Nazism and the terror of fiction [Figure 1] and superimposition of that same fictional fear on the face of the boy who will not be able to differentiate them [Figure 2]: 'Did this universal pain somehow weigh upon the heart of the five-year-old boy who, in the company of his sister -she was seven years older-walked towards the penumbra of a cinema, to see the first film of his life? It is difficult to know.' Then, the narration of that founding experience materialises through a fictional recreation that moves away from the childhood emotion shown in The Spirit of the Beehive. The beam of light from the projector of this B/W recreation gives way to the NO-DO images, on which/while the adult essayist describes the childhood reality in which the trauma is generated: 'This documentary evidence was the substratum of reality on which a few minutes later the fiction would establish itself. But this, the boy didn't know; for him fiction and reality were as yet the same thing.' As Frédérique

Berthet indicates:

The present fear of an imaginary event (the postmen who are messengers of death for the boy) and the fear linked to a past fact (having grazed death under the nationalist bombs for the sister) are therefore activated each other in the darkness of a movie theatre and by the light beam of a Hollywood film (2011: 87).

On the same screen of the fictional recreation of the Kursaal, the film in motion then appears. The images of the first scene of The Scarlet Claw jump to the first screen of the essayistic reflection. The resonance of The Spirit of the Beehive is again inevitable.

Fictional realism made/ensured that the images of Frankenstein were never placed on that first screen, but rather remained framed on the wall of the village cinema. Besides, in opposition to Ana's emotion and the realism of the viewing in the film fiction, Erice chooses a certain representational asepsis for the essay reflection. The archive images of La Morte Rouge are then perfectly in tune with the representation created in The Spirit of the Beehive, while the essayistic recreation abandons the childhood emotion materialised in the fiction film. In other words, while the fictional creation around trauma is generated from the postulates of the realism of representation, the essayistic reflection, once again, leaves that space to materialise from the freedom of hybridisation and experimentation.

The cinematic childhood experience will also star/feature in Erice's second letter to Kiarostami, in which the children of a school in Arroyo de la Luz, Cáceres, see, before the filmmaker's small camera, Kiarostami's film, Where is the Friend's house?/Khaneye doust kodjast? (1987), to be discussed with their teacher later. Unaware of the trauma of that first childhood film experience in the 1940s, sixty years later the children of Arroyo de la Luz identify with the Iranian children, in the same way that Ana did with the little Maria from Frankenstein. Erice then focuses his attention on the other side of childhood experience, that of identification, which makes cinema a magnificent experience of knowledge, as he states in the conclusion of his letter: 'There is a truth that we adults frequently forget: that children do not know borders, that the whole world is their home'. The trauma of the boy Victor is generated on/from this truth, which Erice recreates through the fiction of The Spirit of the Beehive. La Morte Rouge and Goldstadt are also Victor's and Ana's homes respectively. Once again, the diptych structure provides the necessary interstice for reflection, between realist representation and epistemological analysis. While fiction recreates the intimate and subjective childhood experience, modifying the protagonist's behaviour in the face of trauma, who confronts it instead of succumbing to it, the essay film reflects on it, putting it in relation to various elements (historical, sociological, philosophical, cinematographic/cinematic).

The boy then discovers death, and murder, through fiction: 'It was there, in the midst of what the vast majority thought of a/as a mere pastime, that the boy discovered that people died; and, furthermore, that men were able to kill other men.' On the recreated faces of those spectators, now devoid of emotion, the essayist shows the double aspect of trauma: the discovery of death and the pact of silence around it, the secret:

This fact awoke a suspicion in the boy: the unanimous attitude of the adults had to be the consequence of a pact that they had all agreed to, consisting of saying nothing and carrying on watching. Because all of them possessed one feature in common: they knew something he didn't know, a secret that explained everything. To uncover it was, from that moment on, the disturbing adventure to which he felt drawn by a force superior to his will, the one which caused him to glimpse the other face of fiction: a black hole in the fabric of reality, a drain down which the entire innocence of the world had gone.

Thus, Erice reveals how that traumatic childhood experience and the imposed secret would trigger his artistic vocation. He also develops this experience of the secret in The Spirit of the Beehive, generating a fracture in the relationship between father and daughter as a consequence of the relationship that Ana establishes with the maquis finally murdered.

Once the trauma is caused in the cinema, it will accompany the boy as he leaves the screening. Terror has seeped into his real experience, which the adult author then materialises/embodies by means of two new superimpositions: two family photographs next to his sister with the river water flowing at night. In the first, both remain together on the left side of the light beam which, reflected on the water, divides the screen. In the second, this reflection -symbol of the cinematic beam of light-inevitably separates them [Figure 3], as the cinematic experience has done, turning the sister into an executioner: 'He looked into her eyes as if she were a stranger, lips pressed together, saying nothing.' Again through superimposition, Erice generates a new symbolic sentence-image, we witness the thinking in act. The bells of fiction now ring out in the memory of that childhood reality. This same duality is materialised/materialises in The Spirit of the Beehive after the viewing of Frankenstein, when Isabel shows Ana the residence of the spirit and later fakes her own death to scare her [Figure 4]. The real sibling relationship is transformed by trauma. The fiction recreates this transformation and the essay film analyses it, offering an image capable of synthesising it.

The actor-postman-murderer from fiction is transferred to reality. The recreation of cinematic terror as a spectator is now completed with the recreation of real and everyday terror. While it was the terror-image of Holmes and Watson that merged with reality of the piano music played upstairs becomes, in the boy's perception, a new transposition from fiction to reality, from the scarlet claw to the pianist's hand: 'Playing dead was the only way death did wouldn't notice him, as if he were an already claimed victim, and so would pass by without stopping in search of other sleeping bodies. For weeks, the boy lived this nightmare to the full.' Superimposition now uses analogy to materialise/embody the fusion between reality and fiction that occurs in the children's perception [Figure 7]. While Erice analyses here the importance of that claw in trauma, thirty years earlier the filmmaker used it symbolically in the fictional space. In The Spirit of the Beehive, the nightmare turns into a reverie in which Ana faces, trembling, the spirit, and, the menacing claw of the monster also appears in the lower right part of the frame [Figure 8]. Therefore, this fiction image is reinterpreted thanks to the essayistic reflection made possible by the diptych, turning the trauma between reality and fiction into their fraternity. The image of the claw approaching Ana thus stands as a symbolic sentence-image of the catharsis of trauma that fictional creation achieves.

A montage sequence with images of The Scarlet Claw turns the boy spectator into the adult filmmaker who manipulates the original montage, offering an explanation in images of what his voice will then express about the image of the boy:

Until cinema itself came to its aid; if a film had partly perturbed him, there were soon others which little by little placed balm upon the wound. This double game of pain and consolation, of suffering and joy, that reached him from the screen instituted his contradictory relationship with moving images.

The essay film finally offers a synthesis image of the reflection carried out: the light of the cinema projector pierces the image of the five-year-old boy who will become an essential author of Spanish cinema [Figure 9]. The childhood trauma of the origins, the traumatic indiscernibility between fiction and reality, is resolved through a filmic trajectory that first uses fiction in order to achieve the catharsis of trauma, to later transform it into fraternity, in a progressive work of hybridisation that reaches its destination through the essay film in La Morte Rouge.

The Portuguese DVD edition of the film includes a discarded scene, which would be inserted in the middle of the already quoted enunciation (after "balm upon the wound") by means of a cross-fade from that image of the boy pierced by the light of the projector. A new symbolic sentence-image emerges from it: on the beam of light, the posters of the films seen by the boy over the years go over the screen, showing the fictional substratum on which Erice's oeuvre will be built. Among them, the third film that emerges from the beam of light is Frankenstein [Figure 10]. The identification between the young Víctor and Ana is inevitable. Thus, for the last time, the fraternity between reality and fiction generated by the fictional-essayistic diptych is synthesised.

Figure 9. The cinematic beam of light pierces the photo of the boy/young Victor. Figure 10. From that beam, the films that followed the viewing of The Scarlet Claw emerge, among which is Frankenstein.

The absence of this scene in the final montage is thus justified in the text that precedes it:

Discarded montage of a scene from La Morte Rouge for reasons of style. Also, to some extent, for the fear of abounding in the imagery of a certain contemporary cinephilia.

Although more than one title has been included thanks to having its film handout, all of them give an idea of what the cinema of that time represented as popular art.

This is an unfounded fear, in my opinion, since the scene also provides an essential notion for the reflection that concludes, in order to understand the other side of childhood trauma, that of the 'balm, the 'consolation', the 'joy.

Conclusions

'The essayist seeks to know himself, to make/create a discourse on the contemporary by retracing history: in this sense, the essay is the way whereby the individual stands alone in the face of time.' (Thirion, 2015: 444). In La Morte Rouge, through the solitude of the soliloquy, Erice traverses History, and his intimate memory, in order to analyse his first and traumatic cinematic experience. The reflection on the trauma around the indiscernibility between the reality and fiction of childhood perception is materialised/materiaises through different sentence-images of a symbolic nature whose main rhetorical elements are the cross-fade and the superimposition. The essayistic analysis of this traumatic absence of interstice between reality and fiction inevitably invokes The Spirit of the Beehive, in which the filmmaker generated a fictional recreation of the same childhood trauma. A fictional-essayistic diptych is then configured in whose interstice the trauma between reality and fiction becomes fraternity. While the realistic fictional representation of the trauma achieved catharsis and its essayistic reflection materialises/embodies a historical, memory based, filmic and intimate analysis around it, the interstice between the two reveals the bonds that connect both spaces, reality and fiction, transforming the trauma into fraternity. The diptych thus offers a cinematic experience that goes from recreation to analysis, from identification to reflection, from the realist canon to the essayistic freedom, configuring an oscillation in which each film, as an actual image, also becomes a virtual image of the another/other, following the analysis proposed by Deleuze and Parnet: "oscillation, a perpetual exchange between the actual object and its virtual image [...] The actual and the virtual coexist, and enter into a tight circuit which we are continually retracing from one to the other ' (2007: 150). Finally, the diptych also explains Erice's filmic trajectory; from modern to contemporary cinema; from fiction to essay film; from the film industry to the museum experience; from trauma to fraternity.

  ' This excellent conclusion/brilliant ending in the same territory of childhood makes us dream of the gradual construction of Víctor Erice's imagery. It is this fraternity that manages to 'reveal what there may be behind those holes that the action of time opens both in personal memory and in the minutes of History. In conclusion, to highlight the other side of what is sold to us as reality; or what is the same/in other words, to show the other scene' (Erice, 2006: 86).

Monterrubio Ibáñez, L (2018). De un cine epistolar. La presencia de la misiva en el cine francés moderno y contemporáneo, Shangrila Ediciones, Santander. I carry out a study about the use of the letter as intermedial form of the essay film.

the boy's photo before, it is now the image of the fictional recreation of the postman that occupies that place [Figure5]: 'On hearing it, the boy ran to take refuge in the most out-of-the-way corner of the house.' The boy, therefore, does not face fear, does not overcome trauma. The adult filmmaker he becomes manages it through a fiction in which Ana, his alter ego, does not hide from the spirit (Frankenstein-Potts) nor from its transposition into reality (the maquis-the postman), but instead she goes in his search/search of him to find the maquis in the well house [Figure 6] and Frankenstein in her own imagination.

Then, for the first and only time in the film, the screen remains black (36 seconds) while Erice continues his reflection on the experienced fear/ fear he experiences. The absence of image materialises/embodies the paralysis that it causes, the impossibility of action and also of reflection, associated with the words that generate the torment: 'The postman's coming.' It is adult reflection that makes it possible to take up the thinking and, therefore, the image. The fear inflicted by the sister would be a way of exorcising the horror imposed by reality, the one evidenced in the archive images: 'a fear that came from afar, accumulated in the besieged Madrid of the Civil War, during her days as a little girl subjected to the horror of the bombings.' A zoom is then made/The camera zooms in on the image of a girl who recalls the filmmaker's sister, and whose fear, perceptible on her face, would show the fear experienced by the former.

The current adult is capable of giving sense/making sense, through filmic reflection, to/of his sister's behaviour in childhood. In The Spirit of the Beehive we also hear that expression in a nightly conversation between the sisters about the spirit, but in this case referring to the paternal authority: 'Dad's coming, dad's coming,'; a symbol of authority and also of the unspeakable secret. However, Isabel's behaviour is not explained beyond her childhood nature, fiction does not reveal it in relation to historical reality, as the essay experience does.

The recreation of the childhood experience then focuses on the nocturnal experience of the traumatised boy. The successive cross-fades of fixed shots of the house rooms transmit the childhood perception in which, to the indiscernibility between fiction and reality, imagination and oneiric space are now added, where the bells ring again. The


Maximilien Guérin

Louise Esher
email: louise.esher@cnrs.fr.

Jean Léo Léonard
email: jean.leonard@univ-montp3.fr.

Sylvain Loiseau
email: sylvain.loiseau@univ-paris13.fr.

Jean Léo Léonard

Comparing inflectional structure across a dialect group: verb morphology in the 'croissant linguistique'

Keywords: verb inflection, stem spaces, dialect continuum, morphology, dialectology, Croissant linguistique, Gallo-Romance

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

INTRODUCTION 5

Our paper explores how tools developed for the formal modelling of individual inflectional systems may be fruitfully applied to the description of inflection in a dialect group. Traditional dialectology and current theoretical frameworks within inflectional morphology overlap in seeking to account for the phonological realisation of inflectional exponents, and the inventory of functional (morphosyntactic and morphosemantic) categories available in inflection. However, theoretical approaches to inflection additionally seek to capture the structure of the inflectional system itself, by attending to phenomena such as the paradigmatic distribution of exponents and the organisation of lexemes into inflectional classes (see e.g. [START_REF] Stump | Inflectional Morphology[END_REF][START_REF] Stump | Inflectional Paradigms[END_REF][START_REF] Bonami | La structure fine des paradigmes de flexion: Études de morphologie descriptive, théorique et formelle[END_REF]. Consideration of such phenomena is equally of value to dialectological research: formal descriptors of the organisation of inflectional systems provide an illuminating basis for typological comparison [START_REF] Stump | Morphological Typology, From Word to Paradigm[END_REF], including for minority and less-resourced languages, as our paper illustrates.

Our case study focuses on the central Gallo-Romance area traditionally termed Croissant linguistique (i.e. 'Linguistic Crescent', in reference to its roughly crescent-like shape), a transitional zone between Oc and Oïl varieties [START_REF] Tourtoulon | Limite géographique de la langue d'oc et de la langue d'oil[END_REF][START_REF] Ronjat | Essai de syntaxe des parlers provençaux modernes[END_REF][START_REF] Brun-Trigaud | Grammaire structurale du français : le verbe[END_REF]. For each of six survey points located across the Croissant area, we describe the inflectional morphology of the verb, using analytical methods and schematic representations inspired by stem space analysis [START_REF] Bonami | Suppletion and dependency in inflectional morphology[END_REF][START_REF] Bonami | Supplétion et classes flexionnelles[END_REF][START_REF] Bonami | La structure fine des paradigmes de flexion: Études de morphologie descriptive, théorique et formelle[END_REF][START_REF] Bonami | La structure fine des paradigmes de flexion: Études de morphologie descriptive, théorique et formelle[END_REF]. The individual formalisations identify key structural principles of the inflectional system in each variety; comparing these formalisations highlights how these principles group varieties together, and thus provides a genuinely morphological perspective on relations among dialects.

DATA AND METHODS

Data

The data used for our study were elicited in fieldwork interviews conducted between 2016 and 2019 as part of a wider project documenting the varieties of the Croissant (see e.g. [START_REF] Esher | Le Croissant linguistique entre oc, oïl et francoprovençal, Des mots à la grammaire, des parlers aux aires[END_REF], which are today highly endangered. The use and transmission of Croissant varieties has drastically reduced in favour of French, due to a combination of language policy and rural exodus [START_REF] Guérin | Les parlers du Croissant : des parlers minorisés et marginalisés[END_REF]. Currently, the majority of native speakers are aged 70 or over, all speakers are fully bilingual with French, and speakers are sparsely distributed across geographical localities. As well as having limited contact with other speakers, fieldwork informants typically did not perceive their individual varieties as forming part of a wider language group, and thus speaker intuitions about the structure of the dialect continuum were limited to perception of difference between their own variety and any other. The survey points chosen for this study, shown in Figure 1, are Dompierre-les-Eglises (Haute-Vienne), Cellefrouin (Charente), Bonnat (Creuse), Luchapt (Vienne), La Châtre-Langlin (Indre) and Naves (Allier), representing a range of varieties across the Croissant area for which fieldwork data were available. For each survey point, complete inflectional paradigms were obtained for 22 verb lexemes via bilingual elicitation. The fieldwork questionnaire comprised the French items chanter 'sing ', lier 'bind', couver 'incubate', acheter 'buy', aller 'go', blanchir 'whiten', couvrir 'cover', partir 'leave', vendre 'sell', avoir 'have', être 'be', pouvoir 'be able', vouloir 'want', savoir 'know', devoir 'have to', faire 'do', venir 'come', tenir 'hold', dire 'say', croire 'believe', prendre 'take' and voir 'see', selected to illustrate a range of frequent and/or prominent conjugational types occurring in central Gallo-Romance; in practice, all responses were cognate with the French items. Interviews were recorded by the fieldworkers, and our analysis is based on broad IPA transcriptions of the recorded forms. For the purposes of the study, we exclude non-finite forms (i.e. the infinitive and participles) from consideration, as the morphological behaviour of these items is liable to show idiosyncratic divergence from that of finite forms (see e.g. [START_REF] Bach | Morphological evidence for the paradigmatic status of infinitives in French and Occitan[END_REF]. We nevertheless recognise that empirical investigation of the behaviour of non-finite forms constitutes a topic of potential interest and value for future research.

Theoretical analysis

Our analysis is based on the concept of stem spaces [START_REF] Bonami | Suppletion and dependency in inflectional morphology[END_REF][START_REF] Bonami | Supplétion et classes flexionnelles[END_REF], developed for French as a means of visualising the paradigmatic distribution of root allomorphy and the default inheritance relationships between roots. Stem space diagrams identify areas of the inflectional paradigm which are perfectly interpredictable across all lexemes (typically because they share a root), and thus furnish a practical characterisation of the distribution of root allomorphy. Because stem space diagrams focus on exceptionless generalisation, they are not equipped to capture contrasting implicational relationships which are true for subsets of lexemes, although stem graphs or default inheritance trees can be used to visualise relationships between the individual partitions of the stem space (see Bonami 2014 for a critical review of this approach, also [START_REF] Stump | Morphological Typology, From Word to Paradigm[END_REF].

For the purposes of the analysis, we isolate maximally general root, desinential and thematic allomorphs, the distribution of which would be described via blocks of competing rules within a realisational framework such as Paradigm Function Morphology [START_REF] Stump | Inflectional Morphology[END_REF][START_REF] Stump | Inflectional Paradigms[END_REF]. Note that our study is concerned with the paradigmatic distribution of exponents as opposed to sub-word-level relationships between form and function, and thus our segmentation should be understood purely as an abstract, analytical device scaffolding consistent and principled comparison between the inflectional systems examined here: the segmented units are simply phonological substrings.6

THE INFLECTIONAL SYSTEM OF THE VARIETY OF BONNAT

We illustrate our method with reference to the variety of Bonnat. Illustrative paradigms for [ʃɑta] 'sing', [bjɑʃir] 'whiten ' and [dəvre] 'have to' are shown in Tables 1, 2 and 3 respectively: note that stress is phrase-final, as in French, and is thus not pertinent for inflection in synchrony; and that clitic subject pronouns are ordinarily required for finite verb forms in the absence of a full noun subject.

These lexemes represent the three major conjugational types traditionally distinguished for southern Gallo-Romance: the a-type continuing the Latin first conjugation, the i-type continuing the Latin fourth conjugation, and the r-type collapsing reflexes of the Latin second and third conjugations. The exemplar lexemes also illustrate the different extents to which root allomorphy is present: some lexemes, such as [ʃɑta] 'sing', display little or no root allomorphy, while others, such as [dəvre] 'have to', display extensive root allomorphy. Based on comparison of the forms across verbs, the rightmost elements emerge as personal desinences: exponents which are not systematically shared across members of a single TAM category, but which are shared across TAM categories for the same combination of person and number values.

The personal desinences identified for Bonnat are shown in Table 4. On the left are the series of desinences observed in each TAM category; on the right, the three individual series attested in the paradigm overall. As can be seen from these data, there is no variation across TAM categories for the third person singular and all plural persons, while variation in the first person singular is limited to an opposition between /e/ in the future (series γ) and no exponent elsewhere, and variation in the second person singular is limited to an opposition between no exponent in the present indicative and subjunctive (series β), and /ɑ/ elsewhere. The personal desinences are identical for all conjugations. From comparison across lexemes and inflected forms, the leftmost exponents of inflectional forms emerge as roots: exponents which are shared across (a subset of) paradigm cells for the same lexeme, but which are not shared across lexemes (with the exception of the formative [i], [is] in the [bjɑʃir]-type, which will be considered part of the root). Among the consequences of this segmentation is that it isolates certain exponents as neither roots nor desinences. Such exponents will be termed 'thematic elements' or 'stem formatives', and include the sequence [ɔv] found in the imperfect indicative of most 304 lexemes7 ; a formative with context-sensitive variants [i] and [j] found in the imperfect indicative of other lexemes and the conditional of all lexemes; the formative [r] found in the future and conditional; the formatives [e], [er] found in the preterite; and the formative [ɛs] found throughout the imperfect subjunctive. The distribution of thematic elements is discussed in more detail in section 6.

The distribution of roots in the variety of Bonnat is illustrated in Table 5: note that the symbol [ə] is used as in French, to indicate a mid vowel variably realised [ø] or [oe] according to phonological context, but ordinarily deleted; present subjunctive forms for cognates of pouvoir and vouloir were not available; present indicative forms for cognates of être and, in the singular, savoir and avoir, were not segmentable and are given in full. A schematic representation of the distribution of roots is provided in Figure 2, in which phonological forms are replaced by abstract, arbitrary indices (i.e. for a given lexeme, each distinct root is represented by a different letter; there is no conventionalised relationship between any given index and the form of the root it represents).

A B A devoir A B C B D A venir A B C B D A tenir A B C B D A dire A B C A prendre A B C A voir A B C B D A faire A B C B D E croire A B C D E A pouvoir A B C - - D A vouloir A B C - - D A savoir [se/sɑ/sa] A B C D E F avoir [e/ɑ/a] A B C D E être [se/e/e] [sɔ/ate/sɔ] A B C D Figure 2 -Schematic representation of stem distribution in the variety of Bonnat.
The indexical representation in Figure 2 facilitates identification of the distributional generalisations which emerge from the data in Table 5. For example, it is systematically the case that the categories future and conditional share a root, which is ordinarily, though not always, shared by the singular present indicative. Consistently, the plural present indicative forms share a root; all imperfect indicative forms share a root; and these two groups of cells commonly have the same root as each other. Singular present subjunctive forms share a root; plural present subjunctive forms share a root; in most verbs, these two groups of cells have the same root as each other, a root which may be unique to the subjunctive, or, more commonly, shared with the imperfect indicative and plural present indicative. All preterite and imperfect subjunctive cells share a root, excepting in a single lexeme, the cognate of savoir, for which imperfect subjunctive cells have a unique root. Based on these generalisations, we can set up a stem space for this variety as shown in Table 6, in which each numbered zone represents a group of cells which share a root (even if not contiguous); note that the numbers themselves are merely arbitrary indices. The stem space diagram reveals the extent to which contrasts of root allomorphy in the variety of Bonnat map onto morphosemantic feature contrasts between TAM categories, and the morphosyntactic feature contrast between singular and plural forms. As noted in section 2.2, the stem space diagram indicates areas of perfect interpredictability between paradigm cells, which apply across all verb lexemes as nearexceptionless generalisations (the only exceptions occurring within the present indicative forms of isolated, high-frequency lexemes: in Bonnat, the cognates of savoir, avoir and être). The stem space analysis thus focuses exclusively on distributional patterns valid for the inflectional system as a whole, partitioning an idealised inflectional paradigm into indivisible sets of interpredictable cells. In practice, the number of distinct zones identified in the stem space is greater than the number of distinct roots observed for any individual lexeme (for example, the stem space for Bonnat is divided into 8 zones, but individual lexemes exhibit between 1 and 6 distinct roots), and the distribution of a given root typically encompasses two or more zones of the stem space.

The stem spaces set out above are not, in isolation, equipped to capture higher-order paradigmatic generalisations about which zones may share a root within the paradigm of a given lexeme, or finer-grained lexical information about patterns of stem distribution characteristic of subsets of verb lexemes (inflectional classes). Yet attempts to capture such generalisations by means of hierarchical diagrams describing default stem inheritance ultimately prove either inadequate or too powerful (see Bonami 2014 for detailed critique). The problem is magnified for the Croissant dataset under examination here: the dataset contains a relatively low number of individual lexemes, many of which instantiate unique groupings of the stem space partitions. In such a context, few if any reliable generalisations can be drawn about the overall or typical relationships between partitions. For this reason, our analysis focuses instead on the stem space, as an empirically robust basis for comparison between varieties.

COMPARISON OF DESINENTIAL SYSTEMS WITHIN THE CROISSANT AREA

Overview of desinential systems

Comparison of the desinential systems observed (illustrated for the cognate of chanter 'sing' in Tables 7891011)8 reveals a high degree of uniformity in personal desinences for all six varieties. For a given TAM category, the series of personal desinences identified showed no variation across lexemes, and for a given person/number value, variation of exponents across TAM categories is extremely limited. The modern distributions thus contrast markedly with the Latin system from which they have developed, and in which personal desinences vary consistently with inflectional class and TAM categories. The uniformity of personal desinences across lexemes in Croissant varieties argues for the inflectional class system in these varieties being based principally or solely on stem distribution, as proposed by [START_REF] Stump | Morphological Typology, From Word to Paradigm[END_REF] for French (compare also [START_REF] Martinet | De l'économie des formes du verbe en français parlé[END_REF], Dubois 1958).

A further immediately striking property of the Croissant systems is the high incidence of SYNCRETISM [START_REF] Baerman | Syncretism[END_REF]) observed between personal desinences. We hypothesize that the extent of syncretism with respect to person and number values, and the widespread absence of distinctive desinences in singular forms, has been favoured by the obligatory nature of subject pronouns in Croissant varieties.

Paradigmatic distribution of desinences

The distributions observed for personal desinences fall into four distinct groups, corresponding to western, northern, central and eastern areas. The desinence-series, labelled with arbitrary indices α, β, ... are set out in Table 12. The western, northern and central varieties have in common the presence of a consistent series of plural desinences across all TAM categories.

α β α β α β α β γ α β γ α β γ δ ε ζ 1SG - e - ε - ε - - e - -e u i ɔ ɔ - e 2SG - a - ɑ ɑ ɑ ɑ - ɑ ɑ -ɑ aj aj ɑ ɑ ɑ ɑ 3SG - ɔ - ɔ - -- - - - --- -ɔ ɔ - ɔ 1PL
In the western group, a two-way contrast of desinence-series is found in the singular. A unique series of desinences (labelled β in Table 12) occurs in the synthetic future, transparently continuing HABEO 'have.PRS.IND.1SG', HABES 'have.PRS.IND.2SG', HABET 'have.PRS.IND.3SG' respectively, while in all other TAM categories (α), person is marked only by subject clitics and no personal desinences occur, due to the historical deletion of final unstressed vowels in regular sound change.

In the central group, a three-way contrast of desinence-series is found in the singular. Once again, a unique series (labelled γ) occurs in the synthetic future, although in this group the third person singular form of the synthetic future has undergone stress retraction and deletion of the original final vowel, 9 a development characteristic of some Croissant varieties (consider e.g. CANTARE HABET> *[ʃɑtoe'ra] > [ʃɑ'toer] in the variety of Bonnat; likewise DEBERE HABET > [doer], Table 3). In both Bonnat and Dompierre, no distinct personal desinences occur in the present indicative or present subjunctive (series labelled β), again due to regular deletion of final unstressed vowels, as in French; in the other TAM categories, a desinence [ɑ] occurs in the second person singular.

Intermediate between these systems is the more northerly variety of La Châtre-Langlin, with a two-way contrast: the unique series of desinences occurring in the synthetic future (β) is of the type found in Bonnat and Dompierre; in all other TAM categories, the first person singular and third person singular lack a distinct personal desinence, whereas a desinence [ɑ] occurs in second-person singular forms.

Finally, in the variety of Naves, the distribution of personal desinences is more effectively captured not via series of desinences, of which there are six, but via default/exception rules for each individual person/number combination, since for most person/number combinations only two possible exponents occur (excepting the first person singular, for which five possibilities occur). In this variety, the series of exponents found in the present indicative (α) and preterite (β) differ only with respect to the first person singular; the series of exponents found in the imperfect indicative (γ) and synthetic conditional (δ) differ in the first and second person plural; the synthetic future has a unique series (ζ); and only the present subjunctive and imperfect subjunctive share a series in full (ζ).

In all six varieties, the distribution of personal desinences is constant across lexemes: variation is correlated with morphosyntactic and morphosemantic features, but not with arbitrary morphological features such as conjugational class.

THEMATIC ELEMENTS

The principles by which our analysis isolates exponents characterised as roots and personal desinences exclude from consideration certain inflectional formatives occurring between these items, namely 'thematic elements'. The lexical and paradigmatic distribution of such elements is briefly outlined here.10 As will be seen, the groupings which emerge from this overview broadly correspond to the groupings indicated by root allomorphy and desinential systems.

In the preterite (absent in Luchapt), three types of system are found. One, occurring in Bonnat and Dompierre, involves a contrast between two inflectional sequences: [er] in the second person singular and all plural forms, [e] in the first person singular and third person singular forms. Another, found in Naves, is characterised by an exponent [εt] in the second person singular and all plural forms (matching the distribution of [er] in Bonnat and Dompierre), but distinguishes between [ε] in the third person singular and [i] in the first person singular. The third, occurring in Cellefrouin and La Châtre-Langlin, involves a twoway contrast aligned with the singular/plural distinction which is so prominent throughout verb inflection in Croissant varieties: [i] in singular forms, and [ir] (Cellefrouin) or [ij] (La Châtre-Langlin) in plural forms. 310

For the imperfect subjunctive, all personal forms share a distinctive thematic formative: /εs/ in Bonnat and Dompierre, /is/ in Cellefrouin, La Châtre-Langlin and Luchapt. In Naves, imperfect subjunctive forms present a sequence [jas]: while the exponent [as] is unique to the imperfect subjunctive, yod also occurs systematically in a subset of present subjunctive forms (second person singular and all plural forms).

A formative [r] is found throughout the future and conditional forms of all varieties, as is typical for Romance languages. Conditional forms further display a formative realised variously [i], [j] or [ij]. The distribution of these variants is partly a matter of contextsensitive phonology, and partly a matter of morphological specification. In Cellefrouin, Luchapt and La Châtre-Langlin, the contrast aligns with number: [j] occurs in plural forms, while singular forms have [i] (in Cellefrouin and Luchapt) or [ø] (in La Châtre-Langlin). In Bonnat and Dompierre, the opposition is distributed as in the preterite: final [i] in the first and third person singular, and prevocalic [ij] (Dompierre) or [j] (Bonnat) in all other cells. In Naves, the contrast maps not to person but to the phonological shape of individual lexemes' stems: [ij] following a cluster [Cr], [j] following the sequence [Vr].

In some varieties, the conditional and imperfect indicative of all lexemes share a thematic element: this distributional pattern occurs in Luchapt, La Châtre-Langlin and Naves, varieties which ordinarily contrast. Other varieties show a contrast between lexemes in which the conditional and imperfect indicative share a thematic element, and lexemes in which a distinct exponent occurs in the imperfect ([ɑv] in Cellefrouin, [ɔv] in Bonnat, [εv] in Dompierre). Although the contrast originates in an early Romance distinction between first-conjugation and non-first-conjugation lexemes, its modern distribution in the Croissant varieties is no longer readily characterised in terms of individual conjugational types.

The Croissant-internal groupings which emerge here thus correspond to those identified in the discussion of personal desinences: unique forms in the variety of Naves, strong similarity between the systems of Bonnat and Dompierre, and looser resemblance between the systems of Cellefrouin, Luchapt and La Châtre-Langlin.

COMPARISON OF STEM DISTRIBUTION PATTERNS

Illustration of stem distribution patterns

The examples reproduced in Tables 17-22 illustrate the inflected forms of [dəvɛr] 'have to' in the six Croissant varieties under examination; this lexeme is chosen as one which exemplifies typical patterns of stem distribution and points of interest in the data.

Stem distributions are near-exceptionlessly correlated with the morphosyntactic opposition between singular and plural, and with TAM categories. In general, future and conditional forms share a stem; preterite and imperfect subjunctive forms, where attested, share a stem; singular present indicative forms share a stem; and plural present indicative forms share a stem with each other and with all imperfect indicative forms. For the present subjunctive, a single stem is found in all forms in the varieties of Luchapt and Naves, while a singular/plural contrast occurs in the varieties of Cellefrouin, Dompierre and Bonnat. Stem distributions sensitive to person contrasts are rare, limited to distinctive third person singular present indicative forms in Naves. 11 The relationship between these basic distributional blocks varies between the survey points. Where the present subjunctive displays a singular/plural opposition, the plural forms ordinarily pattern with the imperfect indicative and plural present indicative, but the singular forms may be syncretic with the singular present indicative (Dompierre) or have a unique exponent (Cellefrouin, Bonnat). The future and conditional forms share their root with the imperfect indicative and plural present forms in Cellefrouin and Dompierre, but with the singular present indicative forms in Luchapt; while in Naves the synthetic future displays OVERABUNDANCE [START_REF] Thornton | Overabundance (multiple forms realizing the same cell): A noncanonical phenomenon in Italian verb morphology[END_REF], one root being shared with the conditional and the third person singular present indicative, the other with all other paradigm categories. The preterite and imperfect subjunctive, where attested, may have a unique root (Luchapt, Dompierre, Bonnat) or share a root with additional categories including the imperfect indicative (La Châtre-Langlin, Naves).

Stem space diagrams

Combining data from all verbs in the fieldwork questionnaire, we propose the stem space diagrams shown in Tables 2324252627; Bonnat (Table 6) differs from Dompierre (Table 23) only in splitting the preterite and imperfect subjunctive. As illustrated in section 5.1, contrasts of root allomorphy are correlated principally with the morphosyntactic distinction between singular and plural, and with distinctions between TAM categories. Some varieties robustly retain inherited distributional patterns common to other Romance languages: for example, in Cellefrouin and Dompierre, all forms of the preterite and imperfect subjunctive (i.e. surviving reflexes of Latin perfectum forms, Maiden 2001) pattern together, and all forms of the future and conditional (i.e. reflexes of infinitive+'have' periphrases) pattern together. In other varieties, such as that of Naves, both these groupings split along category lines differentiating the preterite from the imperfect subjunctive and the future from the conditional. In the variety of Luchapt, the future and conditional are further fragmented due to an additional singular/plural distinction in some lexemes. A property unique to Naves among the study varieties is the consistency with which the imperfect subjunctive and present subjunctive share an often unique stem, differentiated from the preterite.

In the imperfect indicative, the near-exceptionless tendency is for all person forms to share a stem (a singular/plural split is attested only in La Châtre-Langlin); in the present subjunctive, either all forms share a stem or there is a singular/plural contrast; and in the present indicative, a singular/plural contrast is found in all varieties except that of Naves, where the third person singular form is differentiated from the other five present indicative forms.

The innovation of singular/plural contrasts in TAM categories which do not typically present contrasts in other Romance varieties, and the prevalence of singular/plural contrasts or a uniform root in categories where comparative data create the expectation of a contrast between {1PL, 2PL} and {SG, 3PL}, are characteristic features of the inflectional paradigm of Croissant varieties. Within the Croissant area, this contrast is most developed in La Châtre-Langlin and Luchapt; while the retention of inherited distributional patterns is greatest in Dompierre and Cellefrouin, varieties which, together with Naves, also have the lowest number of distinct partitions of the stem space.

Overall, the stem space analysis reveals greater internal variation than the analysis of desinential and thematic elements: no two varieties display identical stem spaces, and the number of partitions ranges from 6 to 10. Naves is again isolated, lacking the singular/plural contrast and in grouping the two subjunctives into a single partition which contrasts with the preterite; and Dompierre and Bonnat once again cluster closely together. By contrast, La Châtre-Langlin, Luchapt and Cellefrouin diverge considerably from each other: La Châtre-Langlin and Luchapt have each developed unique contrasts, while Cellefrouin, retaining inherited Romance distribution patterns, has the fewest partitions of any variety.

DISCUSSION AND CONCLUSIONS

Our study departs from traditional dialectological practice in focusing on the distribution of inflectional exponents (i.e. the structure of the inflectional paradigm itself) as opposed to the phonological realisation of those exponents (see [START_REF] Weinreich | Is a Structural Dialectology Possible?[END_REF] for a similar approach to phonological contrasts). We compare the organisation of the inflectional system in each different variety, allowing points of similarity and divergence to be readily identified, and providing a genuinely morphological perspective on the internal structure of the dialect continuum.

Our analysis reveals several distinctive tendencies concerning the paradigmatic distribution of exponents in Croissant varieties. The distribution of root allomorphy is becoming increasingly systematised as an exponent of TAM and number: inherited patterns of interpredictability between individual TAM categories are split along category lines, and differentiation within individual TAM categories is aligned with the morphosyntactic contrast between singular and plural. Desinential allomorphy is also more strongly correlated with number than with person, TAM categories or inflectional class, and overall there is pervasive syncretism.

With regard to internal variation, there is a clear typological split, visible throughout the inflectional system, between the isolated variety of Naves, and the other five varieties. Within the larger group, the organisation of desinential elements indicates a central, closeknit group comprising Bonnat and Dompierre; a western group comprising Cellefrouin and Luchapt; and an intermediate group represented by La Châtre-Langlin, which shares some features with each of the other two. The organisation of stem distribution and thematic elements confirms the Bonnat-Dompierre group, but identifies disparities between all three remaining varieties.

Beyond its contribution to description of Croissant varieties, our study exemplifies the value of formal morphological descriptors for the comparison of multiple inflectional systems, and the practicality of such descriptors even in a minority or endangered language context with limited data and considerable variation.

Figure 1 -

 1 Figure 1 -Location of the Croissant within France; survey points within the Croissant.

Table 1

 1 Finite synthetic forms of [ʃɑta] 'sing'

		PRS.IND	IPFV.IND	PRS.SBJV	PRT	IPFV.SBJV	FUT	COND
	1SG	i ʃɑt	i ʃɑtɔv	i ʃɑt	i ʃɑte	i ʃɑtɛs	i ʃɑtre	i ʃɑtri
	2SG	tə ʃɑt	tə ʃɑtɔvɑ	tə ʃɑt	tə ʃɑterɑ	t ə ʃɑtɛsɑ	tə ʃɑtrɑ	t ə ʃɑtərjɑ
	3SG	u/al ʃɑt	u/al ʃɑtɔv	u/al ʃɑt	u/al ʃɑte	u/al ʃɑtɛs	u/al ʃɑtoer	u/al ʃɑtri
	1PL	nə ʃɑtẽ	nə ʃɑtɔvɛ̃	nə ʃɑtɛ̃	n ə ʃɑterɛ̃	nə ʃɑtɛsɛ̃	nə ʃɑtrɛ̃	nə ʃɑtərjɛ̃
	2PL	u ʃɑte	u ʃɑtɔve	u ʃɑte	u ʃɑtere	u ʃɑtɛse	u ʃɑtre	u ʃɑtərje
	3PL	u/al ʃɑtẽ	u/al ʃɑtɔvɛ̃	u/al ʃɑtɛ̃	u/al ʃɑterɛ̃	u/al ʃɑtɛsɛ̃	u/al ʃɑtrɛ̃	u/al ʃɑtərjɛ̃

Table 4

 4 Personal desinences in the variety of Bonnat

		PRS.IND	IPFV.IND	PRS.SBJV	PRT	IPFV.SBJV	FUT	COND	α	β	γ
	1SG	-	-	-	-	-	e	-	-	-	e
	2SG	-	ɑ	-	ɑ	ɑ	ɑ	ɑ	ɑ	-	ɑ
	3SG	-	-	-	-	-	-	-	-	-	-
	1PL	ε̃	ε̃	ε̃	ε̃	ε̃	ε̃	ε̃	ε̃	ε̃	ε̃
	2PL	e	e	e	e	e	e	e	e	e	e
	3PL	ε̃	ε̃	ε̃	ε̃	ε̃	ε̃	ε̃	ε̃	ε̃	ε̃

Table 5

 5 Distribution of root allomorphy in the variety of Bonnat

		PRS.IND.	PRS.IND.	IPFV.IND	PRS.SBJV.	PRS.SBJV.	PRT	IPFV.SBJV	FUT	COND
		SG	PL		SG	PL
	chanter	ʃɑt	ʃɑt	ʃɑt	ʃɑt	ʃɑt	ʃɑt	ʃɑt	ʃɑt	ʃɑt
	blanchir	bjɑʃi	bjɑʃis	bjɑʃis	bjɑʃis	bjɑʃis	bjɑʃis bjɑʃis	bjɑʃi bjɑʃi
	devoir	dø	dəv	dəv	doev	dəv	dədʒ	dədʒ	dø	dø
	venir	vɛ̃	vən	vən	voen	vən	vɛdʒ	vɛdʒ	v ɛ̃	vɛ̃
	tenir	tɛ̃	tən	tən	toen	tən	tɛdʒ	tɛdʒ	t ɛ̃	tɛ̃
	dire	di	diz	diz	diz	diz	dis	dis	di	di
	prendre	prɛ̃	poern	poern	poern	poern	prɛdʒ	prɛdʒ	prɛ̃	prɛ̃
	voir	vø	vəz	vøz	vøz	vəz	vødʒ	vødʒ	vø	vø
	faire	fe	faz	faz	fas	faz	fadʒ	fadʒ	fa	fa
	croire	kre	koerz	koerz	kre	koerj	koerdʒ koerdʒ	kre	kre
	pouvoir	pø	pud	pu	-	-	pudʒ	pudʒ	pø	pø
	vouloir	vo	vul	vu	-	-	vudʒ	vudʒ	vo	vo
	savoir	se/sɑ/sa	sav	sa	saʃ	saʃ	sadʒ	sob	so	so
	avoir	e/ɑ/a	av	a	as	as	adʒ	adʒ	o	o
	être	se/e/e	sɔ/ate/sɔ̃	ɛr	sj	sj	fudʒ	fudʒ	sə	sə

Table 6

 6 Stem space diagram for the variety of Bonnat

		PRS.IND	IPFV.IND	PRS.SBJV	PRT	IPFV.SBJV	FUT	COND
	1SG
	2SG	1		4
	3SG 1PL		3		6	7	8
	2PL	2		5
	3PL

Table 7

 7 Finite synthetic forms of [ʃɑta] 'sing', Cellefrouin

		PRS.IND	IPFV.IND	PRS.SBJV		PRT	IPFV.SBJV	FUT	COND
	1SG	i ʃɑt	i ʃɑtɑv	i ʃɑt		i ʃɑti	-		i ʃɑtre	i ʃɑtri
	2SG	ty ʃɑt	ty ʃɑtɑv	ty ʃɑt		ty ʃɑti	-		ty ʃɑtrɑ	ty ʃɑtri
	3SG	u/a ʃɑt	u/a ʃɑtɑv	u/a ʃɑt		u/a ʃɑti	-		u/a ʃɑtrɔ	u/a ʃɑtri
	1PL	nə ʃɑtɑ̃	nə ʃɑtɑvɑ̃	nə ʃɑtɑ̃		nə ʃɑtirɑ̃	-		nə ʃɑtrɑ̃	nə ʃɑtərjɑ̃
	2PL	və ʃɑte	və ʃɑtɑve	və ʃɑte		və ʃɑtire	-		və ʃɑtre	və ʃɑtərje
	3PL	u/a ʃɑtɑ̃	u/a ʃɑtɑvɑ̃ u/a ʃɑtɑ̃		u/a ʃɑtirɑ̃	-		u/a ʃɑtrɑ̃	u/a ʃɑtərjɑ̃
						Table 8
			Finite synthetic forms of [ʃɑta] 'sing', Luchapt
		PRS.IND	IPFV.IND	PRS.SBJV	PRT	IPFV.SBJV	FUT	COND
	1SG	i ʃɑt	i ʃɑti	i ʃɑt	-	-		i ʃɑtrɛ	i ʃɑtri
	2SG	ty ʃɑt	ty ʃɑti	ty ʃɑt	-	-		ty ʃɑtrɑ	ty ʃɑtri
	3SG	ø/al ʃɑt	ø/al ʃɑti	ø/al ʃɑt	-	-		ø/al ʃɑtrɔ	ø/al ʃɑtri
	1PL	nə ʃɑtɑ̃	nə ʃɑtjɑ̃	nə ʃɑtɑ̃	-	-		nə ʃɑtrɑ̃	n ə ʃɑtərjɑ̃
	2PL	u ʃɑtɛ	u ʃɑtjɛ	u ʃɑtɛ	-	-		u ʃɑtrɛ	u ʃɑtərjɛ
	3PL	i/al ʃɑtɑ̃ i/al ʃɑtjɑ̃ i/al ʃɑtɑ̃	-	-		i/al ʃɑtrɑ̃	i/al ʃɑtərjɑ̃
						16
						Table 9
			Finite synthetic forms of [ʃɑta] 'sing', Dompierre
		PRS.IND	IPFV.IND	PRS.SBJV	PRT		IPFV.SBJV	FUT	COND
	1SG	i ʃɑt	i ʃɑtɛv	i ʃɑt		i ʃɑte		i ʃɑtɛs	i ʃɑtre	i ʃɑtri
	2SG	tə ʃɑt	tə ʃɑtɛvɑ	tə ʃɑt		tə ʃɑterɑ		t ə ʃɑtɛsɑ	tə ʃɑtrɑ	tə ʃɑtrijɑ
	3SG	u/al ʃɑt	u/al ʃɑtɛv	u/al ʃɑt		u/al ʃɑte		u/al ʃɑtɛs	u/al ʃɑtrø	u/al ʃɑtri
	1PL	nə ʃɑtɑ̃	nə ʃɑtɛvɑ̃	nə ʃɑtɑ̃		nə ʃɑterɑ̃		nə ʃɑtɛsɑ̃	nə ʃɑtrɑ̃	nə ʃɑtrijɑ̃
	2PL	u ʃɑtɛ	u ʃɑtɛvɛ	u ʃɑtɛ		u ʃɑterɛ		u ʃɑtɛsɛ	u ʃɑtrɛ	u ʃɑtrijɛ
	3PL	i/al ʃɑtɑ̃	i/al ʃɑtɛvɑ̃	i/al ʃɑtɑ̃		i/al ʃɑterɑ̃		i/al ʃɑtɛsɑ̃	i/al ʃɑtrɑ̃	i/al ʃɑtrijɑ̃
						Table 10
			Finite synthetic forms of [ʃɑta] 'sing', La Châtre-Langlin
		PRS.IND	IPFV.IND	PRS.SBJV		PRT	IPFV.SBJV	FUT	COND
	1SG	i ʃɑt	i ʃɑtø	i ʃɑt		i ʃɑti	i ʃɑtis	i ʃɑtrɛ	i ʃɑtrø
	2SG	ti ʃɑt	ti ʃɑtø	ti ʃɑt		ti ʃɑti	ti ʃɑtis	ti ʃɑtrɑ	ti ʃɑtrø
	3SG	o/al ʃɑt	o/al ʃɑtø	o/al ʃɑt		o/al ʃɑti	o/al ʃɑtis	o/al ʃɑtr	o/al ʃɑtrø
	1PL	i ʃɑtɛ̃	i ʃɑtjɛ̃	i ʃɑtɛ̃		i ʃɑtijɛ̃	i ʃɑtisjɛ̃	i ʃɑtrɛ̃	i ʃɑtərjɛ̃
	2PL	u ʃɑtɛ	u ʃɑtjɛ	u ʃɑtɛ		u ʃɑtijɛ	u ʃɑtisjɛ	u ʃɑtrɛ	u ʃɑtərjɛ
	3PL	i/al ʃɑtɑ̃ i/al ʃɑtjɑ̃	i/al ʃɑtɑ̃		i/al ʃɑtijɑ̃	i/al ʃɑtisjɑ̃	i/al ʃɑtrɑ̃	i/al ʃɑtərjɑ̃

Table 12

 12 Comparison of desinence series in Croissant varieties

	western	northern	central		eastern
	Cellefrouin Luchapt	LaCh.L.	Dompierre	Bonnat	Naves

Table 17

 17 Finite synthetic forms of [dəvɛr] 'have to', Cellefrouin

		PRS.IND	IPFV.IND	PRS.SBJV	PRT	IPFV.SBJV	FUT	COND
	1SG	i dø	i dəvi	i doev	-	-	i dəvre	i dəvri
	2SG	ty dø	ty dəvi	ty doev	-	-	ty dəvrɑ	ty dəvri
	3SG	u/a dø	u/a dəvi	u/a doev	-	-	u/a dəvrɔ	u/a dəvri
	1PL	nə dəvɑ̃	nə dəvjɑ̃	nə dəvɑ̃	-	-	nə dəvrɑ̃	nə dəvrijɑ̃
	2PL	və dəve	və dəvje	və dəve	-	-	və dəvre	və dəvrije
	3PL	u/a dəvɑ̃	u/a dəvjɑ̃	u/a dəvɑ̃	-	-	u/a dəvrɑ̃	u/a dəvrijɑ̃

Table 18

 18 Finite synthetic forms of [dəvɛr] 'have to', Luchapt

		PRS.IND	IPFV.IND	PRS.SBJV	PRT	IPFV.SBJV	FUT	COND
	1SG	i dø	i dəvi	i doej	-	i dəɡis	i dørɛ	i døri
	2SG	ty dø	ty dəvi	ty doej	-	ty dəɡis	ty dørɑ	ty døri
	3SG	ø/al dø	ø/al dəvi	ø/al doej	-	ø/al dəɡis	ø/al dørɔ	ø/al døri
	1PL	nə dəvɑ̃	nə dəvjɑ̃	nə doejɑ̃	-	nə dəɡisjɑ̃	nə dørɑ̃	nə dørjɑ̃
	2PL	u dəvɛ	u dəvjɛ	u doejɛ	-	u dəɡisjɛ	u dørɛ	u dørjɛ
	3PL	i/al dəvɑ̃	i/al dəvjɑ̃	i/al doejɑ̃	-	i/al dəɡisjɑ̃	i/al dørɑ̃	i/al dørjɑ̃

  ; the difference typically concerns root vowel quality, as in Table22, and more rarely affects root-final consonant clusters, as in Table20. For the purposes of the present study, we treat such alternations as a matter of phonology, since they are consistently correlated with alternation between stressed and unstressed roots, and between closed and open syllables. 312

	Table 19 Finite synthetic forms of [dəvɛr] 'have to', Dompierre IPFV.IND PRS.SBJV PRT IPFV.SBJV FUT i dəvɛv i dø i dəɡe i dəɡɛs i dəvre tə dəvɛvɑ tə dø tə dəɡerɑ tə dəɡɛsɑ tə dəvrɑ u/al dəvɛv u/al dø u/al dəɡe u/al dəɡɛs u/al dəvrø nə dəvɛvɑ̃ nə dəvɑ̃ nə dəɡerɑ̃ nə dəɡɛsɑ̃ nə dəvrɑ̃ u dəvɛvɛ u dəvɛ u dəɡerɛ u dəɡɛsɛ u dəvrɛ i/al dəvɛvɑ̃ i/al dəvɑ̃ i/al dəɡerɑ̃ i/al dəɡɛsɑ̃ i/al dəvrɑ̃ Finite synthetic forms of [dəvre] 'have to', Bonnat IPFV.IND PRS.SBJV PRT IPFV.SBJV FUT i dəv i doev i dədʒe i dədʒɛs i dəre tə dəvjɑ tə doev tə dədʒerɑ tə dədʒɛsɑ tə dərɑ u/al dəv u/al doev u/al dədʒe u/al dədʒɛs u/al doer nə dəvjɛ̃ nə dəvɛ̃ nə dədʒerɛ̃ nə dədʒɛsɛ̃ nə dərɛ̃ u dəvje u dəve u dədʒere u dədʒɛse u dəre other person formsTable 20[3] PRS.IND 1SG i dø 2SG tə dø 3SG u/al dø 1PL nə dəvɑ̃ 2PL u dəvɛ 3PL i/al dəvɑ̃ PRS.IND 1SG i dø 2SG tə dø 3SG u/al dø 1PL nə dəvɛ̃ 2PL u dəve 3PL u/al dəvɛ̃ u/al dəvjɛ̃ u/al dəvɛ̃ u/al dədʒerɛ̃ u/al dədʒɛsɛ̃ u/al dərɛ̃ COND i dəvri tə dəvrijɑ u/al dəvri nə dəvrijɑ̃ u dəvrijɛ i/al dəvrijɑ̃ COND i dəri tə dərjɑ u/al dəri nə dərjɛ̃ u dərjɛ u/al dərjɛ̃

Table 21

 21 Finite synthetic forms of [dəvɛr] 'have to', La Châtre-Langlin

		PRS.IND	IPFV.IND	PRS.SBJV	PRT	IPFV.SBJV	FUT	COND
	1SG	i dø	i dəvø	-	i dəvi	i dəvis	i døvrɛ	i døvrø
	2SG	ti dø	ti dəvø	-	ti dəvi	ti dəvisɑ	ti døvrɑ	ti døvrø
	3SG	o/al dø	o/al dəvø	-	o/al dəvi	o/al dəvis	o/al dør	o/al døvrø
	1PL	i dəvɛ̃	i dəvjɛ̃	-	i dəvijɛ̃	i dəvisjɛ̃	i døvrɛ̃	i døvrijɛ̃
	2PL	u dəvɛ	u dəvjɛ	-	u dəvijɛ	u dəvisjɛ	u døvrɛ	u døvrijɛ
	3PL	i/al dəvɑ̃	i/al dəvjɑ̃	-	i/al dəvijɑ̃	i/al dəvisjɑ̃	i/al døvrɑ̃	i/al døvrijɑ̃

See Spencer (2012) for the lack of empirical criteria discriminating between stems and desinences; and[START_REF] Blevins | Word-based morphology[END_REF] for the view that inflectional exponents conventionally referred to as 'stems' and 'desinences' exist not as distinct objects but as abstract generalisations across multiple wordforms.

This formative is etymological in reflexes of the Latin first conjugation, e.g. CANTABAM>[ʃɑtɔv] 'I sang'. In Bonnat, it is undergoing progressive generalisation across verb lexemes, hence the overabundance in the imperfect indicative of [bjɑʃir] (Table2) between forms with etymological yod (<-(I)EBA-) and forms with analogical /ɔv/.

Note that in several varieties the simple past and imperfect subjunctive are no longer in regular use and could not be elicited for all verbs during fieldwork.

At the time of writing, the nature of these changes (regular sound change or analogical innovation) remains to be established.

Analyses assigning thematic elements to stems or desinences could equally be envisaged. Recall that we do not claim any privileged empirical status for our segmentation, which is to be understood as a formal convenience facilitating comparison of inflectional exponents and their distribution.

In the varieties of La Châtre-Langlin (Table22) and Bonnat (Table20[3]), the third person singular future form in some lexemes displays a root phonetically distinct from that occurring in the

We gratefully acknowledge the support of the Agence Nationale de la Recherche (Projet « Les parlers du Croissant : une approche multidisciplinaire du contact oc-oïl», ANR-17-CE27-0001-01), of the Labex EFL (project « Au coeur de la Gallo-Romania : caractérisation linguistique et environnementale d'une aire de transition », ANR-10-LABX-0083) and the project "Oc/Oïl : texts, identity and language contact" funded by the City of Paris. We thank the anonymous RRL reviewer for their constructive comments.


Vincent Le Moigne
email: vincentlemoigne@hotmail.com

Absence of amplification role of the protein KLH on antibody response generated by a MAP Staphyloccocus aureus enterotoxin A (SEA) peptide comparing with the corresponding monomeric peptide

Keywords: Immune response, Peptide, MAP, vaccine

Staphuloccocus aureus enterotoxin A (SEA) is a toxin involved in numerous food poisoning cases. To develop a detection test specific for, different approaches have been envisaged to obtain highest antibody titer sera against a peptide sequence from this protein. The present work compares the properties of antibodies raised against the peptide epitope by classical and multiple antigen peptide (MAP) system approaches. Different immunization protocols were used: BALB/c mice were immunized either with the peptide or the MAP alone in Freund's adjuvant, co-immunized with the keyhole limpet hemocyanin (KLH) protein or, with the peptide, conjugated to this carrier KLH protein.

The extent of reaction of the antibodies to the MAP construct with the parent protein was found to be significantly less than the antibodies raised against the monomeric peptide coimmunized with or conjugated to a carrier protein but more that the antibodies raised against the peptide alone. Inversely, co-immunization of the MAP with the KLH was not able to raise the immune response as it was observed with the monomeric peptide. The results suggest that, for the epitope chosen here, MAP constructs were not the most effective option to induce sera with high levels of antibodies that react with the native protein.

Introduction

Among the wide variety of exoproteins produced by Staphylococcus aureus that contribute to cause disease in mammalian hosts [START_REF] Dinges | Exotoxins of Staphylococcus aureus[END_REF], the S. aureus enterotoxin A (SEA) is of particular importance since 70% of all identified causes of staphylococcal food poisoning are due to SEA [START_REF] Bergdoll | Staphylococcal Intoxications[END_REF]. To elaborate a detection test specific for this protein with antibodies that discriminate SEA from other enterotoxins, a peptide specific of this protein have been selected.

The peptides of about 16-17 amino acids have a molecular weight in the range of 1.7-2 kDa.

These molecular weights are generally not sufficient to evoke an immune response. For this reason, the synthetic peptides must be converted to high-molecular weight products to elicit an antibody response. Branched peptides such as Multiple Antigen Peptides (MAPs) [START_REF] Tam | Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system[END_REF][START_REF] Posnett | A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain[END_REF] have been extensively tested to use single epitope to produce antibodies. MAP consists of a core of branching lysine residues to which peptides epitopes of interest are covalently attached. The resulting polypeptide reaches sufficient molecular weight to stimulate the immune system and could be considered as a small protein. This was an alternative to the classical carrier protein approach consisting to conjugate the peptide to a carrier protein like bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). In addition to obtain a peptide-containing chimeric molecule with high molecular weight, these carrier proteins bring also T-cell epitopes, leading to a proliferation of T-helper cells and an effective stimulation of the immune system. MAP peptides have been used for various immunological applications but mainly for antibodies production and stimulation of the immune system. In this case, they are presented as an efficient alternative to peptide-protein conjugates [START_REF] Tam | Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system[END_REF] with a great potential as immunogens or synthetic vaccines. They have successfully been used for experimental vaccination against various infectious diseases such as malaria [START_REF] Edelman | Immediate-type hypersensitivity and other clinical reactions in volunteers immunized with a synthetic multiantigen peptide vaccine (PfCS-MAP1NYU) against Plasmodium falciparum sporozoites[END_REF][START_REF] Joshi | Immunogenicity of well-characterized synthetic Plasmodium falciparum multiple antigen peptide conjugates[END_REF]Nardin et al., 1995), schistosomiasis (El Ridi et al., 2004) or HIV-1 [START_REF] Misumi | A cyclic dodecapeptide-multipleantigen peptide conjugate from the undecapeptidyl arch (from Arg(168) to Cys(178)) of extracellular loop 2 in CCR5 as a novel human immunodeficiency virus type 1 vaccine[END_REF][START_REF] Nardelli | Cellular immune responses induced by in vivo priming with a lipid-conjugated multimeric antigen peptide[END_REF].

The increased molecular weight and repeating peptide structure of the MAP has been shown to result in improved B-cell recognition and antibody specificity [START_REF] Briand | Application and limitations of the multiple antigen peptide (MAP) system in the production and evaluation of anti-peptide and anti-protein antibodies[END_REF]. MAPs allow also various applications like polyepitopic constructions [START_REF] Posnett | A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain[END_REF], that allow association of peptide of interest with T-cell epitope-containing peptides. Whatever is the case, when comparisons are realized between the peptide either as a monomeric or as a MAP form, only with an adjuvant but without any other protein, the last construction is fully the most efficient to generate the best antibody production [START_REF] Tam | Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system[END_REF][START_REF] Tam | Multiple antigen peptide. A novel approach to increase detection sensitivity of synthetic peptides in solid-phase immunoassays[END_REF][START_REF] Mclean | Generation of anti-peptide and anti-protein sera. Effect of peptide presentation on immunogenicity[END_REF][START_REF] Wang | Long-term high-titer neutralizing activity induced by octameric synthetic HIV-1 antigen[END_REF][START_REF] Francis | Immunological evaluation of the multiple antigen peptide (MAP) system using the major immunogenic site of foot-and-mouth disease virus[END_REF][START_REF] Marguerite | Analysis of antigenicity and immunogenicity of five different chemically defined constructs of a peptide[END_REF][START_REF] Baleux | Novel version of Multiple Antigenic Peptide allowing incorporation on a cysteine functionalized lysine tree[END_REF][START_REF] Marussig | Linear and multiple antigen peptides containing defined T and B epitopes of -19 -the Plasmodium yoelii circumsporozoite protein: antibody-mediated protection and boosting by sporozoite infection[END_REF][START_REF] Farris | Induction of autoimmunity by multivalent immunodominant and subdominant T cell determinants of La (SS-B)[END_REF] However, conclusions are sometimes guarded about efficiency of MAP peptides as good promoters of immune response and some experiments underline the limits of this technique [START_REF] Briand | Application and limitations of the multiple antigen peptide (MAP) system in the production and evaluation of anti-peptide and anti-protein antibodies[END_REF][START_REF] Mahale | Comparison of antibodies raised against the peptide 10-24 of chicken riboflavin carrier protein (cRCP) by classical and multiple antigen peptide (MAP) approaches[END_REF].

In order to estimate and possibly to enhance the effectiveness of antibody production with a multiple antigen peptide, comparisons to immunizations with the peptide without or with the presence of a carrier protein, with or without conjugation, have been realized. Raised antisera in mice were compared in ELISA against the SEA native parent protein or against the peptides (monomeric or MAP).

The administration of the peptide conjugated to KLH induced the best antibody production. Co-immunization of the monomeric peptide with the KLH protein induced a strong increase in antibody production compared with immunization by peptide alone which was the least potent in the induction of such responses. On the other hand, co-immunization of the MAP peptide with KLH did not increase the antibody response.

Materials and Methods

Reagents

The choice of the peptide sequence has been done in order to maximize the probabilities to obtain a good antibody production. In this purpose, well-exposed avoiding complex secondary structures, hydrophilic sequences have been tested. The peptide sequence in this study has been chosen among four other peptides of the SEA protein because it gave the best immune response in preliminary tests on mice. The peptide sequence (TEPSVNYDLFGAQGQYSN) represented the amino acids 190 to 207 of the SEA protein. Peptides were synthesized by the society GenScript corporation (Piscataway, NJ, USA). The monomeric peptide was resupended at 5 mg/ml in deionized water. The same amino acid sequence was synthesized in a multiple antigen peptides (MAPs) form as tetravalent (tetrabranched) construct using FMOC chemistry on an automatic synthesizer according to the manufacturer's protocol. They were then purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and were provided lyophilized and stored desiccated until use. Mass Spectrometry (MS) analysis confirmed the expected molecular mass expected. Resuspension was done at the concentration of 5 mg/ml in deionized water. All experiments reported here were conducted using the same lots of purified peptides.

A second peptide with an added C-terminal cysteine was synthesized allowing conjugation, via the sulfhydryl group of this amino acid, to the amino group of lysine residues on the keyole limpet hemocyanin (KLH) (Sigma, St. Louis, MO, USA) using MBS (m-Maleimidobenzoyl-Nhydroxysuccinimide ester) coupling method. This monomeric peptide conjugation to KLH was effected in order to keep the same peptide orientation as in the MAP.

Highly purified SEA (purity  than 95 %) was obtained from Toxin Technology (Sarasota, FL, USA) and was redisolved in deionized water to a 1 mg/ml concentration.

Animals and immunization

Adult female BALB/c mice (Janvier, Le Genest, France) were divided in groups of 6 mice and immunized with the different formulation of the epitope as a MAP peptide or as a single peptide, either with or without adjunction of KLH as described below and recapitulated in Table 1. For immunization, peptides and MAP were diluted in phosphate-buffered saline (PBS). In each case, the immunizations result in the injection of equal moles of the specific epitope and of KLH when it is present. Two groups were immunized with 25 µg of MAP peptide (corresponding to 10.6 nM of individual peptide sequence) without (group MAP) or with the presence of 125 µg KLH (group MAP + KLH). Two other groups were immunized with 25 µg of the classical peptide (corresponding to 11.7 nM of peptide) without (group Pep) or with the presence of 125 µg KLH (group Pep + KLH). A last group was immunized with 25 µg of the peptide conjugated to the same quantity of KLH as previously (group Pep-KLH). Control mice were immunized with PBS incorporated in Freund's adjuvant for one group and the other group with KLH in Freund's adjuvant.

Mice were immunized at multiple sites by subcutaneously way, four times, with about one month of interval (D0, D28, D57 and D87). Antigens diluted in PBS were emulsified 1:1 in complete Freund's adjuvant (CFA, containing the H37Ra, ATCC 25177 M. tuberculosis strain) for the first immunization (D0) or incomplete Freund's adjuvant (IFA) for the three following immunizations (D28, D57 and D87). Blood samples were collected two weeks after the first immunization (D14) or one week after the last immunization for the boosts (D35, D64 and D94) from the retro-orbital plexus and sera were stored at -20°C until use. All animal experiments were done in accordance with institutional and national ethical guidelines.

Enzyme-linked immunosorbent assay (ELISA)

The antibodies were screened for binding to the monomeric peptide, the MAP and the native protein using the ELISA method. Maxisorp polystyrene microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 0,5 µg/ml of highly purified SEA protein or with 1 µg/ml of monomeric peptide or MAP in 100 µl of carbonate-bicarbonate buffer (0.1 M, pH 9.6).

After a wash with phosphate-buffered saline-Tween 20 (PBS-T) (0.05%, v/v), plates were blocked for one hour at 37°C with PBS-T containing 0.5% bovine serum albumin (PBS-T-BSA). Then serially diluted sera in the same buffer were added to the wells. After 90 min of incubation at 37°C and four washes with PBS-T, goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA alkaline phosphatase-conjugated (Southern biotechnology, Birmingham, USA) were added and the plates were incubated for another 1 h 30 at 37°C. After four washes, 100 µl of 1 mg/ml of p-nitrophenylphosphate (Sigma) in diethanolamine buffer (pH 9.8) was added, and then plates were read at 405 nm with a Titertek Multiscan instrument (Skatron, Oslo, Norway).

Statistical analysis

Significant values were generated using the student t-test. A P value <0.05 was considered to be statistically significant.

-9 -

RESULTS

Antibody titer

Titers of MAP or relative peptide-induced antibodies reactive either with the native SEA protein (Fig. 1), the peptide or the MAP (Fig. 2) were analyzed by ELISA. The results presented here are those obtained after four immunizations but those observed after the second and third immunizations (data not shown) were equivalent in terms of quality.

High titres of antibodies to the parent SEA protein were obtained following the immunization of mice either with the peptide conjugated to KLH or with unconjucated monomeric peptide and KLH (Fig. 1). The titres generated by the MAP formulations were fully weaker either with or without KLH presence. Mice immunized by MAP without KLH presented even better antibody sera titres than mice co-immunized with MAP and KLH. On the contrary response generated by the peptide alone was negligible in comparison with the one generated with peptide KLH-added and with control sera.

Anti-monomeric peptide antibodies generated in presence of KLH bound peptide and MAP almost to the same extent in the ELISA system used (Fig. 2A andB). Conversely, antisera to the MAP construct were more efficient in MAP ELISA system than with monomeric peptide coated ELISA plates. Low titres of antibodies from peptide alone-immunized mice sera were observed in both cases.

After the first immunization (data not shown), only peptide KLH-conjugated immunized mice sera had notable antibody titres when linear monomeric peptide or MAP, but not SEA, were used in ELISA coating.

Antibody isotype and IgG subclass analysis

To further characterize the antibody response, IgG subclass, IgM and IgA reactivities either with, the native S. aureus SEA protein, the peptide or the MAP were examinated by ELISA after the first (Fig. 3) and the fourth immunization (Fig. 4). The optical densities (ODs) obtained for the four IgG subclasses, IgA, and IgM, were measured with sera diluted at 1/625 for the first immunization (Fig. 3) and 1/2500 for the fourth immunization (Fig. 4). The results obtained with the immunoglobulin isotypes are in agreement with those obtained previously in titres study.

The antibody response depended on the formulation of the peptide used during immunization. After the first immunisation (Fig. 3), when the monomeric peptide or the MAP were used for coating (Fig. 3B andC), there was mainly, as expected, weak IgM reponses in mice immunized with the MAP, MAP + KLH, peptide and peptide + KLH. On the contrary, mice immunized with peptide conjugated to KLH already presented a good IgG response, more particularly IgG1 (O.D. = 0.77 vs. peptide and O.D. = 1.89 vs MAP). When SEA was used for coating (Fig. 3A), IgG were not able to recognize the native protein and only a weak IgM response (O.D. = 0.12) was observed in this Pep-KLH group.

After the fourth (third boost) immunizations (Fig. 4), stronger responses were observed in all immunized groups, except group immunized with the monomeric peptide alone. The group immunized with the peptide conjugated to KLH gave the strongest antibody response for IgG1 (O.D. = 3.70), IgG2a (O.D. = 1.59) and IgG2b (O.D. = 2.20) when SEA was used in coating (Fig. 4A). It is interesting to note that this last IgG2b subclass was strongly reduced (P0.0001) when immunization was done with the peptide and KLH unconjugated, while the two other subclasses (IgG1 and IgG2a) presented no significant difference in intensity response. Peptide immunized group did not show any significant antibody response. In comparison to Pep+KLH immunization, the MAP+KLH group response was very weak. For example the strongest immunoglobulin, IgG1, presented a 0.48 O.D. value. The MAP alone immunized group even showed better IgG2a (P0.05) and IgG2b (P0.01) responses than those of this MAP+KLH group.

When the monomeric peptide (Fig. 4B) or the MAP (Fig. 4C) were used in ELISA coating, the results were relatively equivalent regarding the Ig isotype intensity order. As previously, the major fact that could be noticed was the IgG2b response reduction between peptide KLH-conjugated and unconjugated KLH-added immunized mice (P0.002). In addition, some minor observations could be revealed. For example, augmentation of specific IgG3 isotype could be detected in sera from mice when KLH was added to the MAP (P0.001) but only when the monomeric peptide was used for coating (Fig. 4B). With MAP coating (Fig. 4C), there was a weak IgM response with the peptide alone immunization that was not as stronger when using peptide coating (P0.05). (Fig. 4B). Logically, the intensity observed in MAP immunized groups (MAP and MAP+KLH) were globally more important with the MAP coating than with peptide coating.

The results after the second and the third immunizations (data not shown) were equivalent in intensity and antibody isotypes profiles to those observed after the fourth immunization except few minor variations. The results observed in the IgG subclasses and Ig isotypes studies (Fig. 3 and 4) were in agreement with the results reported for titres (Fig. 1 and2).

Globally, an overall view of the results showed that humoral immune response stimulatory potency of peptide plus KLH unconjugated formulation was significantly more efficient than the MAP plus KLH one. This was particularly observed in the parent SEA protein recognition.

Discussion

The main aim in the development of peptide-based antibodies production is to obtain antibodies that recognize the whole original protein. In this purpose, short peptide fragments containing the most potent antigenic determinants can be selected according to physicochemical structural properties such as surface accessibility, hydrophilicity or segmental mobility. However, due to their small size, peptides need to be conjugated to a carrier protein to generate an effective immune response.

Branched immunogens have been developed to bypass the requirement of a carrier and reported to be more stable than linear peptide [START_REF] Fitzmaurice | The assembly and immunological properties of non-linear synthetic immunogens containing T-cell and B-cell determinants[END_REF] and to induce immune response against the peptide branches but not the matrix core, i.e. the polylysine [START_REF] Posnett | A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain[END_REF].

According to [START_REF] Fitzmaurice | The geometry of synthetic peptide-based immunogens affects the efficiency of T cell stimulation by professional antigenpresenting cells[END_REF], peptides branches are presented to T-cells by professional antigen-presenting cells more efficiently than linearly arranged determinants.

Although MAPs are valuable tools for generating specific antibodies, they are often poor immunogens due to their lack of relevant T-cell epitope. To avoid the construction of a chimeric fusion molecule containing an added foreign irrelevant T-cell epitope to the proteic sequence of interest, the goal of this study is to test if co-immunization of the MAP with a complex protein like KLH, is able to improve the immune response.

The MAP construct was immunogenic as shown by the fact that mice elicited high antipeptide antibodies response. Antipeptide antibodies to the MAP construct bound SEA as well as MAP or peptide. As reported elsewhere, when only Freund's adjuvant is used, comparison of immunization between MAP and relative monomeric peptide show without any doubt that MAP are widely more efficient than peptide in specific antibody generation [START_REF] Tam | Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system[END_REF][START_REF] Tam | Multiple antigen peptide. A novel approach to increase detection sensitivity of synthetic peptides in solid-phase immunoassays[END_REF][START_REF] Mclean | Generation of anti-peptide and anti-protein sera. Effect of peptide presentation on immunogenicity[END_REF][START_REF] Wang | Long-term high-titer neutralizing activity induced by octameric synthetic HIV-1 antigen[END_REF][START_REF] Francis | Immunological evaluation of the multiple antigen peptide (MAP) system using the major immunogenic site of foot-and-mouth disease virus[END_REF][START_REF] Marguerite | Analysis of antigenicity and immunogenicity of five different chemically defined constructs of a peptide[END_REF][START_REF] Baleux | Novel version of Multiple Antigenic Peptide allowing incorporation on a cysteine functionalized lysine tree[END_REF][START_REF] Marussig | Linear and multiple antigen peptides containing defined T and B epitopes of -19 -the Plasmodium yoelii circumsporozoite protein: antibody-mediated protection and boosting by sporozoite infection[END_REF]. This is also the case here. However, the extent of binding was found to be significantly less as compared to that of antisera elicited against the peptide-KLH conjugate or peptide co-injected with KLH.

In fact, although the MAP method has been shown to result often in high antibody titers against the peptide, the antibodies seem to recognize less often the protein [START_REF] Mclean | Generation of anti-peptide and anti-protein sera. Effect of peptide presentation on immunogenicity[END_REF][START_REF] Arnon | Structural basis of antigenic specificity and design of new vaccines[END_REF][START_REF] Darcy | Protection of mice and nude rats against toxoplasmosis by a multiple antigenic peptide construction derived from Toxoplasma gondii P30 antigen[END_REF][START_REF] Mahale | Comparison of antibodies raised against the peptide 10-24 of chicken riboflavin carrier protein (cRCP) by classical and multiple antigen peptide (MAP) approaches[END_REF]. One possible explanation of this behavior could be that the polylysine core stabilizes the peptide in a certain conformation and that a less flexible peptide results from this for antibody production.

Concerning the monomeric peptide, conjugation to a carrier protein is not absolutely necessary to obtain serum with high titer level of antibodies but the simple association, via a co-injection, of the peptide with a protein usually used for conjugation, like KLH, is sufficient in the case studied here. This approach of co-immunization could avoid some problems encountered when peptides are conjugated to a protein carrier, resulting often in complex mixture of products. For example, the carrier protein may modify some determinants and the structure of the peptide. However, it does not seem to occur here. Indeed, the best response and parent protein recognition remain when this conjugation is present. In addition, it is interesting to note that conjugation to KLH is a strong inductor of IgG2b in serum.

The surprise comes from the fact that when the MAP is co-injected with KLH, there is not such a huge humoral immune response as those observed for the peptide. On the contrary, there is even a slight decrease in the SEA and monomeric peptide sera recognition capacity in comparison to mice immunized with MAP alone. To explain this, we can refer to works demonstrating that the MAP structure induces T-cell response by itself [START_REF] Olszewska | Protection against measles virus-induced encephalitis by anti-mimotope antibodies: the role of antibody affinity[END_REF][START_REF] Farris | Induction of autoimmunity by multivalent immunodominant and subdominant T cell determinants of La (SS-B)[END_REF][START_REF] Costa | Optimization of an animal model of experimental autoimmune encephalomyelitis achieved with a multiple MOG(35-55)peptide in C57BL6/J strain of mice[END_REF], so, the addition of a protein like KLH is useless to generate T-help.

In addition, it can also be noted that results are similar when the peptide is used with its monomeric or MAP formulation for coating the ELISA plates, even if the MAP coating or -14 -presentation of the peptidic sequence via the MAP is slightly more efficient, as shown by [START_REF] Tam | Multiple antigen peptide. A novel approach to increase detection sensitivity of synthetic peptides in solid-phase immunoassays[END_REF] or [START_REF] Briand | Application and limitations of the multiple antigen peptide (MAP) system in the production and evaluation of anti-peptide and anti-protein antibodies[END_REF]. So, it can be estimated that binding properties to plastic solid surface are relatively the same for the MAP and their monomeric counterparts. As observed in the experiments of [START_REF] Tam | Multiple antigen peptide. A novel approach to increase detection sensitivity of synthetic peptides in solid-phase immunoassays[END_REF] for example, this is not always the case and depends certainly on hydrophobic properties of the peptide. A poly-lysine pre-treatment of the plate could increase the binding of the peptide on the bottom of the plate. Furthermore, it also indicates that antibodies generated against the central lysine core are negligible, in agreement with results obtained in other studies [START_REF] Posnett | A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain[END_REF].

In conclusion, these results demonstrate that the form of the antigen and its association with a protein can profoundly influence the nature and magnitude of the immune response. To obtain good levels of antibody response, the classic method, the simple peptide associated with another protein (with but also without conjugation), remains the best method in the case presented here.

The study of the antibody responses showed that the MAP formulations were not able to induce a good antibody response. The optimal response resulted from the classical method using a carrier protein conjugated monomeric peptide.

Figure 1 :

 1 Figure 1: Reactivities of antisera with SEA by ELISA.

Figure 2 :

 2 Figure 2: Reactivities by ELISA of antisera to monomeric peptide and MAP.

Figure 3 :

 3 Figure 3: Immunoglobulin M, A isotype and Immunoglobulin G subclass analysis after the first

Figure 4 :

 4 Figure 4: Immunoglobulin M, A isotype and Immunoglobulin G subclass analysis after four

Acknowledgements

I would like to thank Wahib Mahana for critical reading of this manuscript.