Nicolas Moulina

François Meurgeyb

Sylvain Hugelc

Julien Chalifour

Mantodea from Eastern Caribbean Islands

Keywords: Lesser Antilles, Caribbean, Mantodea, Acontistidae, Liturgusidae, Thespidae, Epaphroditidae, Mantidae, geographical distribution, ecology

 is revealed to be widely distributed on islands harboring moist and wet forests from Martinique to Guadeloupe. Liturgusa dominica Svenson, 2014 occurs in humid and subhumid forests of Dominica and Marie-Galante. Gonatista reticulata (Thunberg, 1815) and Thesprotia caribea Rehn & Hebard, 1938 appear restricted to the northernmost islands of the Northeast Lesser Antilles. Musonia surinama Saussure, 1869 is present in the grassland of St Martin and of Southern islands (Barbados, St. Vincent, and Grenada). Lobocneme lobipes (Redtenbacher, 1892) is only present in St. Vincent and Grenada. Epaphrodita undulata (Saussure, 1870) is re-established as synonym of Epaphrodita musarum (Palisot de Beauvois, 1805); this species was described from St. Lucia, but has not been recorded for over 150 years. Finally, a specimen of Bantiella trinitatis Giglio-Tos, 1915 was collected on Martinique in 1975, but this species was not collected again. Despite intensive searching, four nonendemic species once recorded on the archipelago have not been recorded again and might correspond to temporary settlements or to erroneous data. Acontista multicolor Saussure, 1870 does not appear to currently occur on Guadeloupe. Distribution, ecology and phenology of these species are discussed. Résumé. Les Mantes des îles de la Caraïbe orientale. Alors que les Mantes de Trinidad et Tobago ont été largement étudiées, les espèces présentes plus au nord et à l'est de l'archipel l'ont beaucoup moins été. L'objectif du présent article est d'actualiser les connaissances sur les mantes du nord-est des Petites Antilles, en nous appuyant sur les données publiées, celles présentes dans la littérature grise, les collections et les spécimens collectés durant les dernières décennies. Thesprotiella insularis (Bonfils, 1967) s'avère largement présent sur les îles arborant des forêts hygrophiles ou mésophiles de la Martinique à la Guadeloupe. Liturgusa dominica Svenson, 2014 est présent en Dominique et à Marie-Galante. Gonatista reticulata (Thunberg, 1815) et Thesprotia caribea Rehn & Hebard, 1938 ont leurs populations restreintes aux îles les plus au nord des Petites Antilles. Musonia surinama Saussure, 1869 est présent dans les milieux herbacés de St. Martin et dans ceux des îles du sud (Barbades, St. Vincent et Grenade). Lobocneme lobipes (Redtenbacher, 1892) est restreint à St. Vincent et Grenade. La synonymie entre Epaphrodita undulata (Saussure, 1870) et Epaphrodita musarum (Palisot de Beauvois, 1805) est rétablie

; cette mante est endémique de St. Lucie, mais n'a plus été signalée depuis plus de 150 ans. Enfin, un spécimen de Bantiella trinitatis Giglio-Tos, 1915 a été collecté en Martinique en 1975 mais n'a plus été signalé depuis. Malgré des recherches intensives, quatre espèces non endémiques, rencontrées une seule fois dans l'Archipel, pourraient correspondre à des établissements temporaires ou à des données erronées. Acontista multicolor Saussure, 1870 ne semble pas se reproduire actuellement en Guadeloupe. La distribution, l'écologie et la phénologie de ces espèces sont discutées.

The Caribbean region is considered a threatened biodiversity hotspot because of its high levels of biotic diversity and endemism and widespread habitat destruction [START_REF] Myers | Biodiversity hotspots for conservation priorities[END_REF][START_REF] Mittermeier | Hotspots Revisited: earth's biologically richest and most endangered terrestrial ecoregions[END_REF][START_REF] Wetterer | Ants of Barbados (Hymenoptera, Formicidae)[END_REF]. A better preservation of this unique biodiversity requires knowing the distribution of the species harbored by these islands. This has been addressed for various groups of insects such as beetles [START_REF] Chalumeau | Les Longicornes des Petites Antilles (Coleoptera, Cerambycidae): taxonomie, Ethologie, Biogéographie[END_REF][START_REF] Peck | The beetle fauna of Dominica, Lesser Antilles (Insecta: Coleoptera): Diversity and distribution[END_REF][START_REF] Ivie | Invertebrates of the assessment of the Center Hills, Montserrat[END_REF][START_REF] Peck | Beetle species diversity in the Lesser Antilles islands: how many species are really there? Insecta Mundi[END_REF][START_REF] Peck | The beetles of the island of St. Vincent, Lesser Antilles (Insecta: Coleoptera); diversity and distributions[END_REF][START_REF] Peck | The diversity and distributions of the beetles (Insecta: Coleoptera) of the northern Leeward Islands, Lesser Antilles[END_REF], 2016; UICN Comité français, OFB, MNHN 2020), moths and butterflies [START_REF] Brown | The origins of the West Indian butterfly fauna[END_REF][START_REF] Miller | The biogeography of the West Indian Butterflies (Lepidoptera): an application of a vicariance/dispersalist model[END_REF]Davies & Birmingham 2002;[START_REF] Fontenla | Biogeography of Antillean butterflies (Lepidoptera: Rhopalocera): patterns of association among areas of endemism[END_REF][START_REF] Pierre | Les papillons de jour de la Martinique (Lepidoptera: Rhopalocera et Castniidae)[END_REF]Touroult et al. 2014a;[START_REF] Zagatti | Catalogue des Lépidoptères des Antilles Françaises[END_REF], and bees [START_REF] Meurgey | Liste préliminaire des abeilles de Guadeloupe (Petites Antilles) et leur relations avec la flore butinée (Hymenoptera: Apoidea:Megachilidae,Apidae)[END_REF][START_REF] Meurgey | Bee species and their associated flowers in the French West Indies (Guadeloupe, Les Saintes, La Désirade, Marie-Galante, St Barthélemy and Martinique) (Hymenoptera: Anthophila: Apoidea)[END_REF][START_REF] Meurgey | Liste préliminaire des Abeilles de Saint-Barthélemy (Antilles françaises) et leurs relations avec la flore butinée (Hymenoptera: Apoidea: Megachilidae, Apidae)[END_REF][START_REF] Meurgey | Les Abeilles de Martinique (Antilles françaises) et leurs relations avec la flore butinée (Hymenoptera: Apoidea: Megachilidae, Apidae)[END_REF][START_REF] Roy | Catalogue du matériel type des mantes conservé au Muséum d'histoire naturelle de Genève (Insecta: Mantodea)[END_REF]; UICN Comité français, OFB, MNHN 2020). Unfortunately, the current decline in biodiversity is accompanied by a growing list of species that are not identified (Touroult et al. 2020a). Knowledge on the Mantises of the Lesser Antilles is very uneven. Whereas species of Trinidad and Tobago at the South of the archipelago are comprehensively known by both historical and recent records (Bruner 1906;[START_REF] Rehn | Orthoptera[END_REF][START_REF] Giglio-Tos | Mantidi esotici. Generi e especie nuove[END_REF][START_REF] Giglio-Tos | Orthoptera Mantidae[END_REF]Beier 1935a;[START_REF] Beebe | An Annotated List of the Mantids (Orthoptera, Mantoidea [sic])[END_REF][START_REF] Crane | A Comparative Study of Innate Defensive Behavior in Trinidad Mantids (Orthoptera, Mantoidea [sic])[END_REF][START_REF] Mcedk | LXXXI. A preliminary list of the Mantodea of Trinidad, B.W.I[END_REF][START_REF] Quesnel | Observations on the reproductive behaviour of the mantis Acontiothespis multicolor[END_REF]Terra 1995;Agudelo et al. 2007;[START_REF] Greener | An Updated List of the Mantodea of Trinidad and Tobago, with Three New Records for Trinidad. Living World[END_REF]Greener & Rutherford 2014), species occurring on more northern islands as well as their distribution are very little known. Nine species are currently recorded from Northeastern Lesser Antilles: Gonatista reticulata [START_REF] Thunberg | Hemipterorum maxillosorum genera illustrate -plurimisque novis speciebus ditata ac descripta[END_REF]; Musonia surinama [START_REF] Saussure | Essai d'un Système des Mantides[END_REF]; Thesprotia caribea [START_REF] Rehn | New genera and species of West Indian Mantidae and Phasmidae -orthoptera[END_REF]; Thesprotiella insularis [START_REF] Bennet | Contribution à l'étude des Orthoptéroides des Antilles. III. Une espèce nouvelle du genre Oligonyx Saussure: description et notes biologiques[END_REF]; Liturgusa dominica [START_REF] Svenson | Revision of the Neotropical bark mantis genus Liturgusa Saussure, 1869 (Insecta, Mantodea, Liturgusini)[END_REF]Acontista multicolor Saussure, 1870; Lobocneme lobipes [START_REF] Redtenbacher | On the Orthoptera of the Island of St. Vincent, West Indies[END_REF]; Bantiella trinitatis [START_REF] Giglio-Tos | Mantidi esotici. Generi e especie nuove[END_REF]and Epaphrodita musarum (Palisot de Beauvois, 1805). Since these early accounts by [START_REF] Thunberg | Hemipterorum maxillosorum genera illustrate -plurimisque novis speciebus ditata ac descripta[END_REF]), Brunner & Redtenbacher (1892), [START_REF] Saussure | Additions au Système des Mantides[END_REF], [START_REF] Rehn | New genera and species of West Indian Mantidae and Phasmidae -orthoptera[END_REF], [START_REF] Bennet | Contribution à l'étude des Orthoptéroides des Antilles. III. Une espèce nouvelle du genre Oligonyx Saussure: description et notes biologiques[END_REF], Mantises were scarcely reported on Northeastern Lesser Antilles in published accounts of scientific surveys [START_REF] Miskimen | The insect fauna of St. Croix, United States Virgin Islands. Scientific Survey of Porto Rico and the Virgin Islands[END_REF][START_REF] Bennet | Contribution à l'étude des Orthoptéroides des Antilles. III. Une espèce nouvelle du genre Oligonyx Saussure: description et notes biologiques[END_REF][START_REF] Woodruff | Checklist and Bibliography of the Insects of Grenada and the Grenadines[END_REF] Recent studied on morphology of many species of Thespidae [START_REF] Rivera | The Neotropical Polymorphic Earless Praying Mantises: a taxonomic review of the genera and checklist of species[END_REF]) allowed transfer of Oligonyx insularis [START_REF] Bennet | Contribution à l'étude des Orthoptéroides des Antilles. III. Une espèce nouvelle du genre Oligonyx Saussure: description et notes biologiques[END_REF] to the genus Thesprotiella. T. insularis is found in Basse-Terre in Guadeloupe, Marie-Galante, Martinique, Dominica [START_REF] Bennet | Contribution à l'étude des Orthoptéroides des Antilles. III. Une espèce nouvelle du genre Oligonyx Saussure: description et notes biologiques[END_REF][START_REF] Bonfils | Catalogue raisonné des insectes des Antilles françaises. 2 Dictyoptera: « Blattaria » et « Mantida[END_REF]Terra 1995;[START_REF] Ehrmann | Mantodeagottesanbeterinnen der Welt[END_REF][START_REF] Otte | Mantida species file. Catalog of the Mantids of the World[END_REF]Agudelo et al. 2007;[START_REF] Meurgey | Liste préliminaire des abeilles de Guadeloupe (Petites Antilles) et leur relations avec la flore butinée (Hymenoptera: Apoidea:Megachilidae,Apidae)[END_REF][START_REF] Patel | Updated Checklist and Distribution of Mantidae (Mantodea: Insecta) of the World[END_REF][START_REF] Meurgey | Challenging the Wallacean shortfall: a total assessment of insect diversity on Guadeloupe (French West Indies), a checklist and bibliography[END_REF][START_REF] Jourdan | Amélioration des connaissances sur les Phasmatodea et les Mantodea de la Guadeloupe et ses dépendances[END_REF][START_REF] Otte | Orthoptéres. Insectes recueillis en Afrique et en Amérique, dans les Royaumea D´Oware et de Benin, a Saint-Domingue et dans les états-unis[END_REF]). An Oligonyx female was also mentioned by [START_REF] Mcedk | LXXXI. A preliminary list of the Mantodea of Trinidad, B.W.I[END_REF] on Trinidad and Tobago, but the specimen was not collected, and Greener & Rutherford (2014) suggested that Kevan confused the specimen with Thesprotia filum. Observations of T. insularis, irrespective of the island, have usually been made in sub humid or rain forests. FM, as part of a study on the Anolis Daudin, 1802 (Squamata, Dactyloidae) of Guadeloupe [START_REF] Legreneur | Distribution des Anolis dans la canopée de la forêt ombrophile en coeur du Parc National de la Guadeloupe et réseaux trophiques[END_REF], was able to access the canopy, between 18 and 25 m high, where he observed many males of T. insularis. These observations appear to confirm the arboreal preference that Bonfils suggested in 1967 and[START_REF] Bonfils | Catalogue raisonné des insectes des Antilles françaises. 2 Dictyoptera: « Blattaria » et « Mantida[END_REF]. The groundlevel observations of this species are likely to occur in individuals that have fallen from higher vegetation. NM has also observed a nymph at 25 m in the crown of a white gum tree (Dacryodes excelsa, Burserarceae), in the heights of the former spa of Absalon in Martinique. T. insularis is a subendemic species of the Lesser Antilles: it is narrowly distributed in an area including part of the Lesser Antilles; with the status 'LC' in the Red Lists of the Fauna of Martinique (UICN Comité français, OFB, MNHN 2020) because it is widespread, and its habitats are not threatened now. [START_REF] Jourdan | Amélioration des connaissances sur les Phasmatodea et les Mantodea de la Guadeloupe et ses dépendances[END_REF]. Its status in Guadeloupe should be published at the end of 2021 by the IUCN. Thesprotia caribea Rehn & Hebard, 1938 (Figures 5a,b,9h,i) Rehn & Hebard 1938, p. 36 [START_REF] Caudell | Report on Orthoptera and Dermaptera collected by the Barbados-Antigua Expedition from the University of Iowa in 1918[END_REF].

Thesprotia caribea
Thesprotia caribea is found on Antigua [START_REF] Rehn | New genera and species of West Indian Mantidae and Phasmidae -orthoptera[END_REF]Terra 1995;[START_REF] Ehrmann | Mantodeagottesanbeterinnen der Welt[END_REF][START_REF] Otte | Mantida species file. Catalog of the Mantids of the World[END_REF]Agudelo et al. 2007;[START_REF] Patel | Updated Checklist and Distribution of Mantidae (Mantodea: Insecta) of the World[END_REF][START_REF] Otte | Orthoptéres. Insectes recueillis en Afrique et en Amérique, dans les Royaumea D´Oware et de Benin, a Saint-Domingue et dans les états-unis[END_REF]). This species was later listed as occurring in "Antigua & Barbuda" without further details [START_REF] Rivera | The Neotropical Polymorphic Earless Praying Mantises: a taxonomic review of the genera and checklist of species[END_REF]). In the area considered in the present work, this species appears restricted to Antigua. Indeed, despite years of surveys, this species is lacking in records from Guadeloupe and Montserrat, respectively the largest and the closest islands around Antigua [START_REF] Jourdan | Amélioration des connaissances sur les Phasmatodea et les Mantodea de la Guadeloupe et ses dépendances[END_REF] and this work for Guadeloupe, [START_REF] Ivie | Invertebrates of the assessment of the Center Hills, Montserrat[END_REF] for Montserrat].

Family Liturgusidae

Liturgusa dominica Svenson, 2014 (Figures 6a,b, Liturgusa dominica was described after specimens collected in the 1960s on Dominica; this description was included in a review [START_REF] Svenson | Revision of the Neotropical bark mantis genus Liturgusa Saussure, 1869 (Insecta, Mantodea, Liturgusini)[END_REF] of the Neotropical genus of bark-mimicking Mantises Liturgusa Saussure, 1869. The species was considered as endemic to Dominica since no other specimens were available. In Marie-Galante, L. dominica occurs in the wettest areas of the sub humid forest around the Saint-Louis river. These are the most humid habitats of the island, which is a raised coral atoll of low elevation. These areas show some similarities with the locality where we observed L. dominica in Dominica. This species can be locally extremely abundant, with several specimens and oothecae present on almost all the trunks of more than 10 cm in diameter. Specimens can be observed both day and night. L. dominica is neither present on the other islands of Guadeloupe (Guadeloupe, Terre-de-Haut and Terre-de-Bas of the Saintes island, the Desirade island), nor in St. Martin, in Martinique, in Saint Lucia, and in Saint-Vincent [START_REF] Touroult | Inventaire entomologique des ZNIEFF de Martinique. Mission 2011 pour le compte de la DEAL Martinique[END_REF][START_REF] Touroult | Inventaire entomologique des ZNIEFF de Martinique et de la Réserve de la Caravelle. Mission 2014 pour le compte de la DEAL Martinique[END_REF]Touroult et al. , 2014b[START_REF] Touroult | Inventaire entomologique des ZNIEFF de Martinique et de la Réserve de la Caravelle. Mission 2014 pour le compte de la DEAL Martinique[END_REF][START_REF] Touroult | Inventaire entomologique des ZNIEFF de Martinique. Campagne de terrain[END_REF]Touroult et al. , 2017Touroult et al. , 2018Touroult et al. , 2019;;[START_REF] Patel | Updated Checklist and Distribution of Mantidae (Mantodea: Insecta) of the World[END_REF][START_REF] Jourdan | Amélioration des connaissances sur les Phasmatodea et les Mantodea de la Guadeloupe et ses dépendances[END_REF]). These islands have been surveyed extensively, as was Montserrat [START_REF] Marske | Effects of volcanic ash on the insect food of the Montserrat oriole Icterus oberi Lawrence 1880[END_REF], where the species is not recorded either. L. dominica is a subendemic species of the Lesser Antilles. In Marie-Galante, its habitats are reduced [START_REF] Joseph | La végétation forestière des Petites Antilles[END_REF][START_REF] Ramade | Impact de l'homme moderne sur les forêts tropicales[END_REF]) even if the populations are large in the known wooded ravines [START_REF] Jourdan | Amélioration des connaissances sur les Phasmatodea et les Mantodea de la Guadeloupe et ses dépendances[END_REF]. Its Red List status in Guadeloupe should be published at the end of 2021 by the IUCN.

Family Acanthopidae Acontista multicolor Saussure, 1870

Acontista multicolor Saussure 1870, p. 229.

Distribution. Guadeloupe, Venezuela, Trinidad & Tobago. Material examined. Guadeloupe. 1♀ Holotype, 'Guadalupe' (MNHN). Despite intensive searching over the last decades, this species has not been found on Guadeloupe. Therefore, Acontista multicolor may be either currently very rare or extinct on Guadeloupe, or the historical mentions may correspond to an erroneous record. Indeed, Saussure (1870) mentions the name of the locality "Guadalupe" which is a very frequent toponym in America, and does not necessarily correspond to the island of Guadeloupe. This species is nevertheless present in Trinidad & Tobago.

Family Epaphroditidae

Gonatista reticulata (Thunberg, 1815) (Figure 9e-g Between the publication of [START_REF] Lombardo | The mantids of Hispaniola, with the description of two species (Mantodea)[END_REF] and that of Agudelo et al. (2007), G. reticulata has been reported from the United States, which seems unlikely. Indeed, the Arizona data in [START_REF] Jantsch | Estudos filogenéticos em Mantódeos americanos (Insecta: Pterygota: Mantodea)[END_REF] was included in 2007 without verification. Later, the distribution of this species is corrected, and its absence is confirmed from the United States [START_REF] Svenson | A Cretaceous-aged Palaeotropical dispersal established an endemic lineage of Caribbean praying mantises[END_REF]. Since its discovery by [START_REF] Thunberg | Hemipterorum maxillosorum genera illustrate -plurimisque novis speciebus ditata ac descripta[END_REF] from "Insula Barthelemi", G. reticulata has never been seen in Saint Barthélemy. Gonatista reticulata is probably present in Saint Barthélemy and well known in Dominican Republic, and Puerto Rico [START_REF] Thunberg | Hemipterorum maxillosorum genera illustrate -plurimisque novis speciebus ditata ac descripta[END_REF][START_REF] Saussure | Essai d'un Système des Mantides[END_REF][START_REF] Caudell | Notes on the mantid genus Gonatista Sauss[END_REF][START_REF] Giglio-Tos | Orthoptera Mantidae[END_REF]Beier 1935b;Terra 1995;[START_REF] Ehrmann | Mantodeagottesanbeterinnen der Welt[END_REF][START_REF] Otte | Mantida species file. Catalog of the Mantids of the World[END_REF]Agudelo et al. 2007;[START_REF] Patel | Updated Checklist and Distribution of Mantidae (Mantodea: Insecta) of the World[END_REF][START_REF] Otte | Orthoptéres. Insectes recueillis en Afrique et en Amérique, dans les Royaumea D´Oware et de Benin, a Saint-Domingue et dans les états-unis[END_REF]INaturalist 2021). The indicated presence in Cuba is an error due to repeated confusion. These specimens from Cuba are G. grisea. Epaphrodita musarum (Palisot de Beauvois, 1805) (Figures 10e,11d) Mantis musarum Palisot de Beauvois 1805, p. 111. Distribution. St. Lucia, Hispaniola. Material examined. St. Lucia: 1♂, Holotype of Epaphrodita undulata, 'Ste Lucia' (MNHN). The single male from St. Lucia was recently considered as a valid species [START_REF] Roy | Epaphrodita undulata (Saussure, 1870) bon species (Dict. Hymenopodidae)[END_REF]. Nevertheless, the morphological diversity of males of E. musarum males from Dominican Republic do not allow it to be distinguished from E. musarum. Three males from Dominican Republic kept in collection (RCNM) were compared to the male labeled from St. Lucia (Table 1). We therefore restore the synonymy established by [START_REF] Giglio-Tos | Orthoptera Mantidae[END_REF]: Epaphrodita undulata [START_REF] Saussure | Additions au Système des Mantides[END_REF] [START_REF] Ippolito | Taxonomic considerations on Lobocneme Rehn, 1911 and Paraoxyopsis Rehn, 1911 and a new synonymy (Insecta Mantodea: Stagmatopterinae)[END_REF]. Lobocneme lobipes is the only mantid of the Mantidae family present in Northern Lesser Antilles. The species is known in Grenada and St. Vincent [START_REF] Redtenbacher | On the Orthoptera of the Island of St. Vincent, West Indies[END_REF][START_REF] Brunner Von Wattenwyl | On the Orthoptera of the Island of Grenada, West-Indies[END_REF][START_REF] Saussure | Fam. Mantidae. In Biologia Centrali-Americana. Insecta Orthoptera[END_REF][START_REF] Jourdan | Amélioration des connaissances sur les Phasmatodea et les Mantodea de la Guadeloupe et ses dépendances[END_REF][START_REF] Rehn | Orthoptera[END_REF][START_REF] Giglio-Tos | Mantidae esotici. VII. Vatinae[END_REF][START_REF] Giglio-Tos | Orthoptera Mantidae[END_REF][START_REF] Marshall | Catalogue of the primary types of Mantodea in the British Museum (Natural History)[END_REF]Terra 1995;[START_REF] Ehrmann | Mantodeagottesanbeterinnen der Welt[END_REF][START_REF] Otte | Mantida species file. Catalog of the Mantids of the World[END_REF]Agudelo et al. 2007;[START_REF] Ippolito | Taxonomic considerations on Lobocneme Rehn, 1911 and Paraoxyopsis Rehn, 1911 and a new synonymy (Insecta Mantodea: Stagmatopterinae)[END_REF][START_REF] Patel | Updated Checklist and Distribution of Mantidae (Mantodea: Insecta) of the World[END_REF][START_REF] Otte | Orthoptéres. Insectes recueillis en Afrique et en Amérique, dans les Royaumea D´Oware et de Benin, a Saint-Domingue et dans les états-unis[END_REF]. The male holotype of L. lobipes is illustrated in a recent publication by [START_REF] Ippolito | Taxonomic considerations on Lobocneme Rehn, 1911 and Paraoxyopsis Rehn, 1911 and a new synonymy (Insecta Mantodea: Stagmatopterinae)[END_REF]. The female allotype from Grenada is illustrated in a publication by Brunner von Wattenwyl (1893). FM reportedly observed L. lobipes specimens in rain forests of St. Vincent without collecting them. The genus Lobocneme is close to Parastagmatoptera. The latter is known to be mainly arboricolous in French Guyana [START_REF] Roy | Catalogue du matériel type des mantes conservé au Muséum d'histoire naturelle de Genève (Insecta: Mantodea)[END_REF][START_REF] Moulin | Synthèse des connaissances des Mantodea de Guyane[END_REF]. L. lobipes could plausibly occur in sub humid and rain forests.

Discussion and conclusion

We recorded nine species of Mantodea on Eastern Caribbean Islands. The islands have neither been connected in the past nor have they been connected to the continent. Southern Lesser Antilles emerged around 15-20 million years ago and the Northern Lesser Antilles emerged more recently, around 5-1.25 million years ago [START_REF] Liebherr | Zoogeography of Caribbean insects[END_REF][START_REF] Wetterer | Ants of Barbados (Hymenoptera, Formicidae)[END_REF][START_REF] Crews | Towards a synthesis of the Caribbean biogeography of terrestrial arthropods[END_REF]. Therefore, all terrestrial species occurring on these islands result from at least one event of colonization [START_REF] Rosen | A vicariance model of Caribbean biogeography[END_REF]. Four species are only found on these islands and should therefore be considered as endemic to this part of the Caribbean: Thesprotiella insularis, Thesprotia caribea, Liturgusa dominica and Lobocneme lobipes (Table 2). Three endemic species (T. insularis, L. dominica and L. lobipes) are not restricted to one single island but occur on nearby islands in the island arc. Interestingly, all of these endemic species display an arboricolous ecology and are all found in sub humid and rain forests; except T. caribea, which occurs in herbaceous areas. The five other species occur on other islands of the Caribbean (Gonatista reticulata, Epaphrodita musarum) or are more widely distributed in Central America (Musonia surinama, Bantiella trinitatis, Acontista multicolor). A. multicolor, E. musarum, G. reticulata and B. trinitatis are arboricolous species, whereas M. surinama occurs in more herbaceous areas. These four species with wide distribution occur more in dry habitats compared to the habitat of the three endemic species (T. insularis, L. dominica and L. lobipes). While the distribution of endemic species is restricted to adjacent islands in the arc, non-endemic species are either known from one single island or from islands scattered on the archipelago. It is worth mentioning that the four non-endemic species known from one single island (Acontista multicolor, Epaphrodita musarum, Bantiella trinitatis and Gonatista reticulata) are only known from one single record and have not been collected again (Table 2). These scarce observations may either correspond to temporary settlements and/or to species occurring at very low density. It might also correspond to under sampling, particularly for T. caribea observed only in Antigua in 1918, E. musarum only in St. Lucia before 1870, like A. multicolor in Guadeloupe, and for G. reticulata the rediscovery of which is pending on tree trunks of St. Barthélemy. One of the non-endemic species, Musonia surinama, is discontinuously distributed, occurring on the southernmost and a few northern-most islands (Table 2). Curiously, this species is lacking from large islands on the middle of the arc. Yet, some of these islands apparently display large rather dry herbaceous habitats suitable for the species, particularly the southern part of Martinique and the eastern part of Guadeloupe. The discontinuous distribution of Musonia surinama in the arc may be explained by various colonization scenarios, involving distinct colonization waves, including one from the south as it has been shown for other groups [START_REF] Brown | The origins of the West Indian butterfly fauna[END_REF][START_REF] Woods | The fossil and recent land mammals of the West Indies: an analysis of the origin, evolution, and extinction of an insular fauna. Biogeographical Aspects of Insularity[END_REF]Hedges 1996aHedges , 1996b;;[START_REF] Miller | The biogeography of the West Indian Butterflies (Lepidoptera): an application of a vicariance/dispersalist model[END_REF]Davies & Birmingham 2002;[START_REF] Fontenla | Biogeography of Antillean butterflies (Lepidoptera: Rhopalocera): patterns of association among areas of endemism[END_REF]. It may also involve more recent introduction in one area from the other one. It is possible that tropical storms carried timber with oothecae or even live specimens from island to island. A species of the genus Mantoida has been observed in captivity preferably depositing its oothecae in small holes directly in the wood. Such crevices occur in the wild, for example as exit holes of other insects, such as saproxylic beetles [START_REF] Wieland | Unrecognized museum specimen expands distribution of Mantoida (Insecta: Mantodea) into the central Caribbean[END_REF]. The transfer of Mantodea oothecae via driftwood has previously been described [START_REF] Werner | Bemerkungen über die geographische Verbreitung der Mantodeen (Fangheuschrecken)[END_REF][START_REF] Werner | Verschleppung von Fangheuschrecken (Mantodeen) durch den Schiffer-Verkehr[END_REF][START_REF] Beier | Die geographische Verbreitung der Mantodeen. Internationaler 7[END_REF][START_REF] Ehrmann | Mantodeagottesanbeterinnen der Welt[END_REF][START_REF] Harris | Living Statilia maculata Thunberg (Insecta: Mantodea: Mantidae) and other invertebrates, frequently imported into Dunedin on used cars[END_REF]. Oothecae of bark mantises (L. dominica, G. reticulata) and arboricolous species (T. insularis, L. lobipes) are usually attached to branches, on tree trunks [START_REF] Svenson | Revision of the Neotropical bark mantis genus Liturgusa Saussure, 1869 (Insecta, Mantodea, Liturgusini)[END_REF][START_REF] Rivera | The Neotropical « polymorphic earless praying mantises" -part I: molecular phylogeny and revised higher-level systematics (Insecta: Mantodea, Acanthopoidea)[END_REF][START_REF] Brannoch | Manual of praying mantis morphology, nomenclature, and practices (Insecta, Mantodea)[END_REF] or deposited on the underside of leaves. Therefore, the dispersal of Mantodea via hurricanes and driftwood carrying oothecae or insects would provide a plausible explanation [START_REF] Clark | Notes on the insects of Barbados, St. Vincent, The Grenadines, and Grenada[END_REF]. Wind dispersal of insects is a well-known phenomenon that may have played a role in some species such as B. trinitatis [START_REF] Wiktelius | Wind dispersal of insects[END_REF]Pasek 1988). However, species dispersions can also be due to human trade, accidental introductions [START_REF] Harris | Living Statilia maculata Thunberg (Insecta: Mantodea: Mantidae) and other invertebrates, frequently imported into Dunedin on used cars[END_REF]. For example, 8% of the entomofauna in Guadeloupe is considered introduced by industry, agriculture, and trade [START_REF] Meurgey | Challenging the Wallacean shortfall: a total assessment of insect diversity on Guadeloupe (French West Indies), a checklist and bibliography[END_REF]. The same goes for Barbados [START_REF] Clark | Notes on the insects of Barbados, St. Vincent, The Grenadines, and Grenada[END_REF][START_REF] Nutting | Barbados-Antigua expedition; narrative and preliminary report of a zoological expedition from the University of Iowa to the Lesser Antilles under the auspices of the Graduate College[END_REF]. Alternatively, to scenarios involving colonization events, this discontinuous distribution with areas of occurrence separated by large suitable habitats may result from local extinctions [START_REF] Wunderle | From the past to the globalized future for Caribbean birds[END_REF]. Indeed, the very habitat of Musonia surinama may have been in competition with past farming practices on these large islands, and insectivorous species such as mantids are likely to be directly and indirectly impacted by past use of pesticides [START_REF] Krooss | The effect of different farming systems on epigeic arthropods: a five-year study on the rove beetle fauna (Coleoptera: Staphylinidae) of winter wheat[END_REF][START_REF] Clermont-Dauphin | Effects of intensive monocropping of bananas on properties volcanic soils in the uplands of the French West Indies[END_REF][START_REF] Cabidoche | Long-term pollution by chlordecone of tropical volcanic soils in the French West Indies: a simple leaching model accounts for current residue[END_REF][START_REF] Crabit | Soil and river contamination patterns of chlordecone in a tropical volcanic catchment in the French West Indies (Guadeloupe)[END_REF]. Such speculative explanation linked to farming practices may also be consistent with the current lack of endemic mantid species on the herbaceous habitats of islands such as Guadeloupe and Martinique. On the other hand, agricultural practices can neither fully explain the lack of these species on central islands of the arc, since open forest and edges may be able to harbor graminicolous species too. Although a large proportion of these islands is protected (over 60%), most of the protected areas are covered by forest [START_REF] Gould | First Records of Mantids (Insecta: Mantodea) on Huevos Island, Trinidad and Tobago. Living World[END_REF]. Ongoing molecular studies may help to explain the current distribution of endemic and non-endemic mantids on these islands.

  (CDR); 1♀, Montagne du Vauclin, Vauclin, 21.IV.2020, Romé leg. (CDR); 1♀, Les Anses d'Arlet, Morne Bigot, 12. VIII.2020, Ravat leg. (CPR); 1 nymph, Fort-de-France, Station thermale Absalon, 25 m Canopy in Dacryodes excelsa (Burseraceae), 7.X.2020, Moulin leg. (RCNM); 1 nymph, Le Marigot, 25.X.2020, Dubreuil leg. (CTD); 1 nymph, Fonds Saint-Denis, Morne Piquet, 15.XII.2020, Viscardi leg. (CMC). Other data. Dominica. 3♂, S. Chiltern, 5.XI-10. XII.1964, Spangler leg. (USNM) (Rivera & Svenson 2020); 1♂, Trois Pitons, 22.X.1964, Spangler leg. (USNM) (Rivera & Svenson 2020); 2♂, 1.3 mi. E of Pont Casse, 12.V-27.IV.1964, Flint leg. (USNM) (Rivera & Svenson 2020); Nymphs, Alford Estate, Chopin, 20.XII.1966, Bonfils & Jean-Bart leg. (Bonfils 1967); 1♂, St. Paul Springfield State 2.5 km ENE Canefield 15°21ʹN, 61°22ʹW, 450 m, 11-18.VI.1991, Rawlins & Thompson leg. (CM); 1♂, I.1999, Montgomery leg. (ANSP) (Rivera & Svenson 2020).

)

  Liturgusa dominica Svenson 2014, p. 110. Material examined. Marie-Galante. 1♂, 1♀, Saint-Louis, Les Sources, 2.VIII.2007, Hugel leg. (CSH); 3♂, 1♀, 1 nymph, Saint-Louis, Les Sources, 6.VIII.2011, Hugel leg. (CSH); 2♀, Saint-Louis, Les Sources, 7. VIII.2017, Hugel leg. (CSH); 2♂, Capesterre de Marie Galante, Les Balisiers, 6.VIII.2017, Hugel leg. (CSH); 1 nymph, Saint-Louis, Ravine Poirier, 29.V.2018, Leblond leg. (CFM); 4♂, 3♀, Saint-Louis, Les Sources, hand catching, 4-5.VI.2019, Moulin leg. (RCNM); 3♂, 1♀, Capesterre de Marie-Galante, Les Balisiers, hand catching, 5.VI.2019, Moulin leg. (RCNM). Dominica. 1♂, 2♀, 1 nymph, Spanny falls trail, 26. VII.2008, Hugel leg. (CSH, ABS). Other data. Dominica. 1♀, Paratype, Fond Figues, 16. III.1964, Bray leg. (USNM) (Rivera & Svenson 2020); 1♂, Holotype, 1♀, Allotype, 1 nymph and 3♂, Paratypes, Grand Bay, -15°14'22.32"N 61°19'12.36"W, Flint leg. (USNM) (Rivera & Svenson 2020); 1 nymph, Paratype, Clarke Hall, 22-31.X.1966, Gurney leg. (USNM) (Rivera & Svenson 2020); 1♀, Paratype, B.W.I., La Haut Estate, 18.I.1979, Finnamore leg. (Lyman Museum) (Rivera & Svenson 2020).

)

  Mantis reticulataThunberg 1815, p. 288. Distribution. Saint Barthélemy, Dominican Republic, Puerto Rico. Other data. Saint Barthélemy. 1♂ Holotype (Thunberg 1815).

  ). Refuge des 3 Crêtes, 2.V.2020, Jourdan leg. (CTJ); 1♀, Bouillante, Duprat, D14, 25.V.2020, Tremblay leg. (CRT). Marie-Galante. 1♀, Grand Bourg, Les Sources, VIII.2004, Hugel leg. (CSH); Grand Bourg, Les Sources, 2.VIII.2007, Hugel leg. (CSH); 1♂, 2♀, 1 nymph, 1 ootheca, Grand Bourg, Coulée Ouliée haut, 8.VIII.2011, Hugel leg. (CSH); 1♀, Capesterre de Marie-Galante, Les Balisiers, 6.VIII.2017, Hugel leg. (CSH); 1♀, Capesterre de Marie-Galante, les Balisiers, hand catching, 5.VI.2019, Moulin leg. (RCNM); 1♂, Saint-Louis, Les Sources, LED trap, 6.VI.2019, Moulin leg. (RCNM); 1 nymph, Saint-Louis, Ravine Potens, hand catching, 6.VI.2019, Moulin leg. (RCNM).

	Martinique. 1♂, Morne Rouge, Périnelle, 17.I.1954, Pinchon leg. (CRPP); 3♂, Gros Morne, Annexe, 27.
	IV.1955, Pinchon leg. (CRPP); 1 nymph, Colson, 5. VI.1966, Pinchon leg. (CRPP); 1♂, Gros Morne, Bonfils
	leg. 1969 (MNHN); 1♀, Trinité, 4.XI.1975, Pinchon leg. (CRPP); 1♂, Sainte-Marie, 2.XII.1975, Pinchon leg.
	(CRPP); 1♂, La Médaille, Fort-de-France, 19.I.1977, Pinchon leg. (CRPP); 1♂, Marigot, Morne Laroche, 30.
	III.2008, David leg. (CGD); 1♂, Fonds Saint-Denis, Arborétum de l'Alma, 3.VIII.2008, Hugel leg. (CSH); 1♂,
	Morne Vert Pré, Le Vert Pré, VI.2010, Delannoye leg. (CRD); 1♀, Route forestière de Grand Fond, Fond Saint-
	Denis, 20.IX.2010, Deknuydt leg. (CFD); 1 nymph, Piton du Carbet, 1.III.2011, Romé leg. (CDR); 1♀, Route
	forestière de Grand Fond, Fond Saint-Denis, 12.IX.2011, Delannoye leg. (CRD); 1♂, Morne Bellevue, Le
	Marigot, 10.IV.2012, Deknuydt leg. (CFD); 1♀, La Montagne du Vauclin, Le Vauclin, 22.IV.2012, Romé leg.
	(CDR); 1♀, La Montagne du Vauclin par Ensfelder, Le Vauclin, 24.IV.2012, Delannoye leg. (CRD); 1♀,
	Maison du Moine, 26.VII.2012, Delannoye leg. (CRD); 1 nymph, La Montagne du Vauclin par Ensfelder, Le
	Vauclin, 11. II.2014, Delannoye leg. (CRD); 1♀, Forêt de l'Epinay, 26.II.2014, Romé leg. (CDR); 1♀, Morne
	Bigot, Les Anses d'Arlet, 15.XII.2014, Ravat Ph. (CPR); 1♂, Morne Constant, Le Diamant, 10.X.2015, Touroult
	& Poirier leg. (RCNM); 1♂, Morne Gentil, Les Anses d'Arlet, 13.X.2015, Touroult & Poirier leg. (RCNM); 1
	nymph, Rivière Rouge, Gros Morne, 26.IV.2016, Delannoye leg. (CRD); 1♂, Rivière Rouge, Malaise Trap, 2-9.
	XI.2017, Dumbardon-Martial leg. (CEDM); 1♂, Morne Sulpice, 24.XI.2017, Moulin & Ramage leg. (RCNM); 1
	nymph, Morne-Rouge, 3.IV.2019, Touroult leg. (RCNM); 1♂, Le François, Morne Valentin, 28.IV.2019, Poirier
	& Touroult leg. (RCNM); 1♀, Rivière Pilote, Forêt de Lépinay, 22.XII.2019, Romé leg.

Family Mantidae Lobocneme lobipes Brunner & Redtenbacher, 1892 (Figure 7, b)

  , synonym of Epaphrodita musarum (Palisot de Beauvois, 1805). Lobocneme lobipes Brunner & Redtenbacher 1892, p. 206. 1♂, Holotype, south end of island, open swampy land near sea, beaten from bushes, 27.IX.1891, Smith leg. (BM) (Redtenbacher 1892); 1♀, St. Vincent, 30. VIII.1902, Campbell leg. (Rehn 1905); 1♂, Kingstown, 15. V.1937, Lombardo leg.

	Distribution. St. Vincent, Grenada.
	Other data. Grenada. 1♀, Mount Gay Estate, Granville (Windward side), -III.1893 (Brunner von Wattenwyl
	1893).
	St. Vincent.

Acknowledgements

We would like to thank all of the contributors for their data and photographs that allowed this work to update the distributional knowledge of Mantodea from the northern Lesser Antilles (in alphabetic order): Julien Chalifour, Donatien Charles, Mathieu Coulis, Gwénael David, Francis Deknuydt, Régis Delannoye, Thomas Dubreuil, Eddy Dumbardon-Martial, Mathieu Jegu, Toni Jourdan (and work into the field), Gilles Leblond, Pierre-Damien Lucas, Eddy Poirier, Marc Pollet, Karl Questel, Philippe Ravat, Daniel Romé, Julien Touroult, Romain Tremblay, Guillaume Viscardi. We thank Philippe Grandcolas, Frédéric Legendre and Roger Roy for access to the Mantodea collections of MNHN; as well as the outstanding work of Mantodea Species File website managers. Sampling in the canopy in Martinique was made possible thanks to the association "Tout Là-Haut" represented by Matias Loubes. Field surveys were partly supported by the Muséum national d'Histoire naturelle, Paris and Parc National de la Guadeloupe (convention 2016-SJ416-16). We thank the National Parks and Forestry Services of Guadeloupe, Dominica, Martinique, St. Lucia and St. Vincent for giving access and allowing research in the protected areas. In Guadeloupe, we thank the Parc National de Guadeloupe, the DEAL Guadeloupe, and particularly Claudie Pavis and Anne-Marie Toussaint (INRAE, Guadeloupe) for access to INRAE campus in Guadeloupe, Hervé Magnin, Louis Redaud and Guy Van Laere. In Dominica, SH thanks the Dominica Forestry, Wildlife & Parks Division, particularly James Arlington and Albert Gallion; SH also thanks the team of Archbold Tropical Research Center, Springfield Field Station Dominica, particularly Nancy Osler for assistance. In Martinique, SH and NM thanks the DEAL Martinique, particularly Julien Mailles, Stephan Lerider and Julie Gresser, and Parc Naturel Régional de la Martinique, particularly Bénédicte Chanteur. In Saint Lucia, SH thanks The Forestry Department and particularly Michael Bobb; SH thanks also Lorraine Royall and Melvin Smith for their kind help. In St. Vincent, SH thanks The Forestry Department and particularly Fitzgerald Providence; SH also thanks Mark de Silva for his precious advices. NM thanks Lena Baraud, Arzhvaël Jeusset and Florian Kirchner for the rigorous work of assessing insect species within the framework of the IUCN Red Lists in the French Antilles. We thank Antoine Mantilleri (MNHN) and two anonymous reviewers who made valuable comments on earlier drafts of the manuscript.


Frank Dietsch

Yves Nominé

Audrey Stoessel

Camille Kostmann

Anna Bonhoure

Bruno Chatton

Mariel Donzeau
email: mariel.donzeau@unistra.fr

Small p53 derived peptide suitable for robust nanobodies dimerization

Keywords: V H Hs, bivalency, dimerization domain, peptide, p53, avidity

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

INTRODUCTION

Nanobodies (V H Hs) are valuable tools for various applications, which are directly linked to their small size, outstanding stability and solubility, and their capacity to fold in a reducing environment [START_REF] De Meyer | Nanobody-based products as research and diagnostic tools[END_REF][START_REF] Dumoulin | Single-domain antibody fragments with high conformational stability[END_REF][START_REF] Muyldermans | Nanobodies: natural single-domain antibodies[END_REF]. For example, two innovative developments for research are chromobodies as tracers for in vivo intracellular targets and immunomodulators to interfere in vivo with protein functions or localization [START_REF] Rothbauer | Targeting and tracing antigens in live cells with fluorescent nanobodies[END_REF]. For diagnostic, V H Hs labeled with a radionuclide are applied as detection tracers [START_REF] De Meyer | Nanobody-based products as research and diagnostic tools[END_REF].

V H Hs are monovalent, possessing only one paratope binding capacity. However, multivalency, which is a general nature of antibodies, increases the apparent affinity compared to monovalent molecules for binding to "repeated" epitopes or to surface-bound antigens [START_REF] Donzeau | Recombinant monoclonal antibodies[END_REF][START_REF] Pack | Monoand Bivalent Antibody Fragments Produced in E. coli: Binding Properties and Folding in vivo[END_REF][START_REF] Rheinnecker | Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen[END_REF]. Therefore, bivalency might be advantageous or even crucial for particular applications. Converting a V H H into bivalent format can dramatically increase its potencies and binding capacities when compared to the related monovalent counterpart [START_REF] Beirnaert | Bivalent Llama Single-Domain Antibody Fragments against Tumor Necrosis Factor Have Picomolar Potencies due to Intramolecular Interactions[END_REF][START_REF] Coppieters | Formatted anti-tumor necrosis factor α VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis[END_REF][START_REF] De | Single-Domain Antibodies and Their Formatting to Combat Viral Infections[END_REF][START_REF] Klooster | Selection and characterization of KDEL-specific VHH antibody fragments and their application in the study of ER resident protein expression[END_REF]. Indeed, an engineered bivalent V H H directed against the trimeric envelope protein of the Respiratory Syncytial Virus (RSV) neutralized the RSV Long strain about 4000-fold better than the monovalent format [START_REF] Hultberg | Llama-Derived Single Domain Antibodies to Build Multivalent, Superpotent and Broadened Neutralizing Anti-Viral Molecules[END_REF], and the conversion of a human TNF-specific V H H binder into bivalent format enhanced its neutralizing potency up to 400-fold (from nM to pM range) [START_REF] Coppieters | Formatted anti-tumor necrosis factor α VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis[END_REF].

. Bivalent formats are generally achieved by using linkers of various length generally introduced by genetic engineering, creating V H H in tandems. DNA sequences coding either for hinge regions composed of glycine and serine residues or for the natural IgG2c hinge of llama heavy-chain antibodies, are inserted between the two V H H coding sequences [START_REF] De | Single-Domain Antibodies and Their Formatting to Combat Viral Infections[END_REF][START_REF] Conrath | Camel Single-domain Antibodies as Modular Building Units in Bispecific and Bivalent Antibody Constructs[END_REF][START_REF] Rothbauer | Targeting and tracing antigens in live cells with fluorescent nanobodies[END_REF] producing a tail-to-head fusion format. Another way of managing bivalency is the addition of a dimerization tag or domain, such as GCN4 or IgG Fc [START_REF] Cardoso | Single-Domain Antibodies Targeting Neuraminidase Protect against an H5N1 Influenza Virus Challenge[END_REF][START_REF] De Meyer | Nanobody-based products as research and diagnostic tools[END_REF][START_REF] Saerens | Single-domain antibodies as building blocks for novel therapeutics[END_REF] in the C-terminal domain of the V H H, given rise to a tail-to-tail format. The last way is by chemical conjugation [START_REF] Van Lith | Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates[END_REF]. All three methods have been used successfully.

We have recently taken advantage of a modified tetramerization domain of p53 to construct bifunctional and bispecific heterotetramer biomolecules [START_REF] Stoessel | Modular Conjugation of a Potent Anti-HER2 Immunotoxin Using Coassociating Peptides[END_REF][START_REF] Vigneron | Self-Associating Peptides for Modular Bifunctional Conjugation of Tetramer Macromolecules in Living Cells[END_REF]. This tetramerization domain (p53tet) is a small peptide of 31-aminoacids, which can be fused to various moieties. In this work, we use the mutant p53tet-E343K/E346K (hereinafter referred to as E3), where two lysine residues were mutated in glutamic acid residues, enabling to exclusively form an homodimer extremely stable in vitro and in cellulo [START_REF] Brokx | A Stable Human p53 Heterotetramer Based on Constructive Charge Interactions within the Tetramerization Domain[END_REF]. We demonstrate that compared to tandem linked bivalent V H H, such a homodimer discloses the same and even improves biophysical properties in term of solubility, avidity and stability in living cells.

MATERIAL AND METHODS

Cell Lines

U2OS, H2B-eGFP HeLa cells and MRC5 YFP-polη cells were maintained as monolayer in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/L glucose, 10 % fetal calf serum (FCS) and 1 mM pyruvate. Cells were transiently transfected with expression plasmids using JetpEI reagent (Polyplus transfection, Illkirch) according to the manufacturer's protocol or electro-transfected with proteins as previously described [START_REF] Freund | Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells[END_REF]. elsewhere [START_REF] Baldeck | FF483-484 motif of human Pol mediates its interaction with the POLD2 subunit of Pol and contributes to DNA damage tolerance[END_REF][START_REF] Freund | Generation of an intrabody-based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells[END_REF][START_REF] Masson | Preferential nuclear localization of the human papillomavirus type 16 E6 oncoprotein in cervical carcinoma cells[END_REF][START_REF] Shima | Partitioning of the Golgi apparatus during mitosis in living HeLa cells[END_REF][START_REF] Vigneron | Self-Associating Peptides for Modular Bifunctional Conjugation of Tetramer Macromolecules in Living Cells[END_REF]. The doubled-stranded DNA sequence oligonucleotide encoding a mitochondriatargeting signal (MTS) MISLTARLSRSAPDSQACYSV-LGAFLGKCADV was cloned between the HindIII and the PstI sites of pEGFP-N1 vector.

Recombinant plasmid constructions

DNA

Expression and purification of the recombinant fusion proteins

Briefly, eGFP, avitag-eGFP, and the nanobodies were expressed in E. coli BL21 (DE3) pLysS by induction with 1 mM IPTG at 20 °C overnight. Proteins were purified on HiTrap IMAC resin (1 ml) saturated with nickel or cobalt (GE Healthcare Saclay, France) and subsequently loaded on HiLoad 16/600 75 prep grade column (GE Healthcare, Bio-sciences AB, sweden) operating at a flow rate of 0.5 ml/ min. Fractions were separated on SDS-PAGE gels and analyzed by Coomasie blue.

Biotinylation of avitag-eGFP

Typically, enzymatic biotinylation was performed with 50 mM ATP (Adenosine triphosphate); 10 mM Magnesium Acetate; 150 µM d-biotin; 6 µg BirA enzyme added to 500 µg purified avitag-eGFP in solution in PBS x1. The mix was incubated for 48 h at 4°C and the biotinylated product was purified by SEC. Biotinylation efficiency was evaluated by incubating the biotinylated protein with saturating amount of streptavidin magnetic beads (GE Healthcare, Bio-sciences AB, sweden), thus, all biotinylated proteins bind the streptavidin and the amount of remaining proteins can be estimated by loading the supernatant on SDS-PAGE.

Enzyme-linked immunosorbent assay (ELISA)

Indirect ELISA, which usually involves two binding processes of primary antibody and secondary labeled antibody was modified as follow: Maxisorp microtiter plates (Nunc, Denmark) were coated with 20 µg/ml of recombinant PCNA in phosphate buffer, PBS, pH 8, for 16 h at 4 °C. Wells were blocked with 0.2 ml of 2% (w/v) milk in PBS, pH 7.4, containing 0.1% (v/v) of Tween-20 for 1 h at RT. A dilution series of purified tagged proteins was added to the pre-coated microtiter plates beginning with a concentration of 10 µM and incubated ON at 4 °C. After extensive washing with PBS, recombinant proteins were detected with a secondary c-myc 9E10 mouse antibody followed by a ternary peroxidase conjugated antimouse antibody (Dianova, Germany) at 2 µg / ml. The signal was developed by addition of 100 µl / well of TMB color reagent (sodium acetate 0.1 M, pH 6.6, 0.1 mg / ml of 3, 3', 5, 5'tetramethylbenzidine (TMB), 2 µl of 35% Hydrogen peroxide, and stopped with 50 µl of sulfuric acid at 0.45 M. The signal was monitored with an ELISA plate reader (BioRad 550) at a wavelength of 450 nm. Data were first processed with the dose-response curve (drc) package of the R language using the following four-parameter log-logistic function to determine the

apparent K D : f (x) = Min + Max -Min 1+ K D x ⎛ ⎝ ⎜ ⎞ ⎠ ⎟
b , where x is the concentration.

Surface Plasmon Resonance

Protein-protein interaction assays were performed on a Biacore T200 instrument (Cytiva, Malborough, MA, USA) at 25 °C using PBS buffer pH 7.4 supplemented with surfactant polysorbate 20 (Cytiva) 0.005% (v/v) as running buffer. The biotin CAPture kit was used as previously described [START_REF] Bonhoure | One-step affinity purification of fusionproteins with optimal monodispersityand biological activity: applicationto aggregation-prone HPV E6 proteins[END_REF], allowing the reverse capture of the biotinylated eGFP antigen. Briefly, in each cycle, a 5-fold dilution solution of the CAPture reagent is injected on all four channels for 300 s at 2 µL/min. One channel (harboring only CAPture reagent and no biotinylated peptide) was systematically defined as the reference flow cell to serve as a control for non-specific binding of the analyte. Biotinylated eGFP was immobilized by injecting a 100 nM solution for 30, 60 or 90 s contact time at 15 µL/min on distinct cells.

One of the three nano-eGFP constructs was then injected as analyte on the four channels for seconds. At the end of each cycle, the surface was fully regenerated by injecting the regeneration solution (GdmCl 6 M, NaOH 0.25 M) as indicated by the manufacturer.

Kinetic measurements were achieved by injecting a series of two-fold concentration of nano-eGFP constructs, in the 0.12 -1000.0 nM range, by increasing concentration order. The 2.0 and 62.5 nM concentrations of the nano-eGFP constructs were systematically replicated at the end of the concentration series in order to assess for reproducibility. All the assays were recorded for 3 different eGFP immobilization levels (roughly ~60, ~180 and ~260 RU) related to the three different ligand contact time. Data for the shortest contact time, and therefore the lowest immobilization signal of eGST protein (~60 RU), were considered for analysis in order to reduce the binding capacity of the surface and minimize the possible mass transport effects.

Data were first processed using the Biacore T200 Evaluation 1.0 software (Cytiva) for correcting for the buffer effects and bulk refractive index changes, followed by an in-house

Python script for automatically superimposing and dividing each sensorgram by the theoretical maximum normalized binding signal R max,norm . R max,norm was determined according to [START_REF] Karlsson | Practical aspects concerning direct detection of low molecular weight analytes using BIACORE[END_REF]:

where MW analyte and MW ligand are the molecular weights of the analytes and the biotinylated eGFP ligand (29.5 kDa), respectively, R immob is the immobilization level of the biotinylated-eGFP ligand. Here we assumed that the ligand owns one analyte binding site per ligand molecule and that the ligand is 100% active on the surface. Such a processing allows to directly compare sensorgrams obtained for monomers (nano-eGFP-mono and nano-eGFPtandem) or dimers (nano-eGFP-E3).

Immunofluorescence

Cells grown on coverslips were fixed with 4% (w/v) paraformaldehyde for 30 min and then permeabilized with 0.2% Triton X-100 for 5 min. In some experiments, the cells were treated with ice-cold cytoskeletal buffer (CSK: 10 mM HEPES pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA) with 0.2% Triton-X100 for 5 min prior fixation. Cells were either observed by direct immunofluorescence or further incubated with the purified recombinant proteins in PBS at different concentrations. The recombinant proteins were then detected with the anti-c-Myc 9E10 monoclonal antibody (1/5000) followed by anti-mouse antibody conjugated with Alexa Fluor 568 (1/10000) (Life Technologies). Coverslips were mounted with Fluoromount G containing 4',6'-diamidino-2 phenyleindole (SouthernBiotech, Birmingham, UK). The treated cells were analyzed using conventional fluorescence microscopy with a Leica DM5500 microscope (Leica Microsystems, Wetzlar, Germany).

Images were processed with Image J2.0.0.

RESULTS:

R max,norm = MW analyte MW ligand R immob
We use the well-characterized nanobody directed against the green fluorescent protein, eGFP, (cAbGFP4) referred herein as nano-eGFP-mono that has been described to have a K D measured by SPR of 0.32 nM [START_REF] Saerens | Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies[END_REF]. Bivalency was achieved either by genetically fusing the nano-eGFP coding sequence in a tandem with a spacer composed of four glycine residues followed by one serine repeated 3 times (hereinafter referred as to nano-eGFP-tandem), or via the self-associating peptide E3 (hereinafter refereed as to nano-eGFP-E3), which originates from the tetramerization domain of p53 (residues 325-355) (Figure 1A) [START_REF] Vigneron | Self-Associating Peptides for Modular Bifunctional Conjugation of Tetramer Macromolecules in Living Cells[END_REF]. The paratopes of these two bivalent nanobodies have a different orientation relatively to each other, namely a tail-to-head and a tail-to-tail orientation, respectively. The structure of the tetramerization domain of p53 is depicted according to PDB1C26 [START_REF] Jeffrey | Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms[END_REF]. The DNA sequences encoding each module are represented in the left panel (light green box for nano-eGFP, gray box for a GGGGS linker repeated 3 fold and dark green box for the E3 helix). The theoretical oligomerization states of the corresponding proteins with the expected molecular weights of each monomeric species are depicted on the right panel.

Recombinant proteins were expressed in E. coli. The nano-eGFP-mono, nano-eGFPtandem and nano-eGFP-E3 were produced at high levels (around 80 mg/ L) and were highly soluble with no notable difference between all three constructs (Figure S1). The three nanobodies were purified on immobilized metal affinity chromatography followed by a preparative size exclusion chromatography (Figure S2). Typical elution profiles revealed that the nano-eGFP-mono and the nano-eGFP-tandem behaved both as monomers, while the Bivalency usually increases the apparent binding constant to surface-bound antigen.

Thus, an indirect ELISA experiment was performed against a plastic-immobilized recombinant eGFP (Figure 2A). The nanobodies were detecting the eGFP, yet with different efficiencies.

The apparent binding efficiencies of the dimers nano-eGFP-E3 and the nano-eGFP-tandem were moderately enhanced by about 10-and 7-fold as compared to the nano-eGFP-mono, respectively. Thus, dimerization in both cases leads to a modest gain of binding efficiency likely by means of avidity. Furthermore, the nano-eGFP-E3 and the nano-eGFP-tandem exhibit a fitted slope coefficient of the Hill function larger than one (b = 1.23 ± 0.07 and 1.13 ± 0.07) as compared to a b value for the nano-eGFP-mono below one, (b = 0.74 ± 0.12), which is suggesting a positive cooperativity of the bivalent format for the surface-bound antigen (Table S1) [START_REF] Syedbasha | An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions[END_REF].

Biosensor Surface Plasmon Resonance (SPR) experiments were conducted to compare the binding kinetics of the three nanobodies for the eGFP-antigen immobilized on a CAP sensor chip (Figure 2B). Although none of the steady state or kinetic fits can be performed on the sensorgrams, kinetic traces revealed several interesting aspects of the interaction between the eGFP and the V H H nanobodies. During the post-injection phase, the signal was almost constant over the time, suggesting a possible strong affinity but impeding binding kinetic rates estimation. Extending the dissociation time at a delay as long as 20 min did not lead to a significant lost of signal. Furthermore, curves obtained for the various concentrations of the different constructs remained almost parallel, suggesting the absence of significant visible differences on the dissociation rates k off of the three constructs. During the injection phase, the equilibrium state was not reached at the end of the phase for low analyte concentrations, impeding a steady-state analysis. Yet, such an analysis would have been interesting in order to compare with the indirect ELISA data, which are recorded at equilibrium due to the inherently long incubation time. Meanwhile, the theoretical maximal normalized response R max,norm (1.00) was reached for nano-eGFP-E3, although it was not attained for nano-eGFP-mono (plateau at R norm ~0.9) for unclear reasons. The difference with R max,norm was even more pronounced for the nano-eGFP-tandem (plateau at R norm ~0.7) as compared to the nano-eGFP-E3 homodimer. Nevertheless, the differential effect of the V H Hs can be explored by comparing normalized sensorgrams obtained for a same concentration. A 2.0 nM concentration value, in addition to belong to the range of apparent affinities determined by indirect ELISA assay, seems to be a good compromise to avoid high concentration values for which the sensorgrams display significant differences in curvature, and low values for which the signal increase is limited. With about 25% signal increase, the sensorgram comparison revealed a possible moderate gain of apparent affinity for nano-eGFP-tandem and nano-eGFP-E3 compared to the monomeric nano-eGFP-mono, confirming the results obtained by the indirect ELISA assay. analyte / ligand molecular weight ratio, and assumes a 1:1 binding model (by considering here the nano-eGFP-E3 as a homodimer, and the nano-eGFP-tandem and nano-eGFP-mono as monomeric species, each of them possibly interacting with a single eGFP partner). Such normalized sensorgrams allow to directly compared signal obtained for different valencies. Kinetic runs were performed by injecting series of two-fold cascade dilution of the nano-eGFP constructs between 0.12 and 1000 nM, in increasing order, i.e. a total of 14 distinct concentrations. Repeated sensorgrams (at 2.0 or 62.5 nM) do not reveal visible differences, confirming the good reproducibility of the assay. Sensor surface is regenerated after each cycle. The thin dotted black line corresponds to the theoretical maximal normalized response (1.00), while the dashed red line corresponds to the Rnorm signal attained by the nano-eGFP-E3 at the end of the injection phase.

The binding efficiencies of the nano-eGFP constructs were next evaluated by conventional immunofluorescence detection after fixation and permeabilization of the HeLa cells stably expressing the H2B-eGFP tagged protein [START_REF] Rothbauer | Targeting and tracing antigens in live cells with fluorescent nanobodies[END_REF] (Figure 3). At eGFP with a better efficiency for the bivalent formats. At 0.8 nM, only the nano-eGFP-E3 was able to detect the H2B-eGFP. This result was unexpected as both constructs displayed very similar apparent affinity at equilibrium. Moreover, one should however note that at the 4 nM, while the nano-eGFP-E3 could clearly detect the chromatin-bound antigen, the nano-eGFPtandem detection already triggered a slight background increased. The ability of the three different nano-eGFP formats to bind to eGFP fusion proteins inside of living cells was then investigated on distinct eGFP fusion proteins localized in different cell compartments. (i) The β-galactosidase-eGFP either with a nuclear localization signal (NLS) enabling its nuclear transport or without, and therefore restricted to the cytoplasmic compartment was evaluated [START_REF] Masson | Preferential nuclear localization of the human papillomavirus type 16 E6 oncoprotein in cervical carcinoma cells[END_REF]. (ii) Two eGFP-fusion proteins embedded in the chromatin consisting of the polymerase-η fused to YFP [START_REF] Baldeck | FF483-484 motif of human Pol mediates its interaction with the POLD2 subunit of Pol and contributes to DNA damage tolerance[END_REF] and a chimera (P40M1-eGFP-con1) binding strongly to the proliferating cell nuclear antigen (PCNA), a processivity factor of the replicative polymerases, were tested [START_REF] Freund | Generation of an intrabody-based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells[END_REF].

(iii) Finally, the eGFP directed to the mitochondria compartment using a mitochondria-targeting sequence (MTS-eGFP) was also used.

The first strategy consisted to transiently co-transfect expression plasmids encoding the nanobodies and the eGFP-antigens in U2OS cells (Figure 4AB). The nanobodies were revealed using a monoclonal anti-c-Myc antibody followed by a fluorescent anti-mouse antibody and the eGFP-antigen was visualized by direct immunofluorescence. All three nano-eGFP formats were able to bind to the eGFP fusion proteins, yet with different binding efficiencies. Monovalent and bivalent formats were clearly sequestered in the cytoplasm through their binding to β-galactosidase-eGFP, while the negative control, nano-Lamin, was freely diffusing into the nucleus, and thus distributed in both compartments (Figure 4A).

Conversely, both bivalent V H Hs were distributed exclusively in the nucleus when co- transfected with eGFP-NLS-β-Galactosidase, while the monovalent were slightly detected in the cytoplasm (Figure 4A).

To detect the chimera (P40M1-eGFP-con1), which strongly interacts with PCNA tightly bound to the chromatin [START_REF] Moldovan | PCNA, the Maestro of the Replication Fork[END_REF], cells were treated or not with cytoskeletal buffer (CSK) prior fixation (Figure 4B and Figure S3). CSK treatment removes freely and loosely held proteins in order to reveal only PCNA molecules at replication foci. Without CSK treatment, as the amount of the V H Hs is exceeding the chromatin-bound PCNA, it was not possible to clearly detect PCNA foci (Figure S3). However, with CSK treatment, the foci corresponding to chromatin-bound PCNA were then revealed by all three nanobodies while with different efficiencies (Figure 4B). Indeed, the nano-GFP-E3 and the nano-GFP-tandem were distinctly better at detecting PCNA foci than the nano-GFP-mono, leading to high background level.

Finally, as the MTS-eGFP is a soluble antigen, no CSK treatment could be applied.

The detection by all three nanobodies was not successful and led to a blurry signal (Figure S4). Nevertheless, these results confirmed the similar avidity effect of both bivalent nano-eGFP in regard to the monovalent counterpart. Because transfection of expression plasmids drives an overload / excess of nanobody compared to the antigen, and because CSK treatment is not appropriate to eliminate unbound V H Hs to freely mobile antigens, a second strategy consisting to directly electro-transfer the purified nanobodies was implemented. In that case, the nanobodies are generally present at lower levels than the target antigen.

For the H2B-eGFP antigen, the expression of the eGFP target antigen was high enough to titrate the three transduced nanobodies (Figure 5A

A B

obvious detection differences between the monovalent and the bivalent formats. These interactions were specific, as the nano-HER2 used as a negative control [START_REF] Stoessel | Modular Conjugation of a Potent Anti-HER2 Immunotoxin Using Coassociating Peptides[END_REF], did not bind to H2B-eGFP and thus localized in both cytoplasmic and nuclear compartments. For the MTS-eGFP localized in the mitochondria compartment, the transduced V H Hs clearly co-localized with the eGFP signal except for the negative control nano-HER2, as expected (Figure 5A right panel). The nano-eGFP-mono led to higher background compared to the bivalent formats.

Lastly, the translesional DNA polymerase-η fused to YFP (YFP-polη) was also used as an antigen bound to the chromatin. The Polη plays a pivotal role to bypass replication arrest following DNA lesions induced by ultraviolet irradiation and thus accumulates at DNA foci through its direct interaction with PCNA. The YFP-polη expressing stable cell lines were electro-transferred with the three purified nanobodies (Figure 5B and 5C). Then, MRC5 cells were UV-irradiated 24 h after transfection and treated without or with CSK buffer 3 h later, before fixation. Without CSK treatment, the two bivalent V H Hs were able to detect YFP-polη, but a sharper signal was given by the nano-eGFP-E3 (Figure 5B). The nano-GFP-mono yielded to a high level of non-specific signals. After CSK treatment, the signals given by all nanobodies were better delineated because the excess of non-bound V H Hs was washed out to reveal those tightly bound to YFP-polη (Figure 5B). Statistic analyzes demonstrate the superiority of both bivalent V H H over the monovalent format (Figure 5C). Indeed, around 85% of the YFP-polη foci signal co-localized with the signal of the nano-eGFP-E3, and 65% for the nano-eGFP-tandem, while less than 30% for the monovalent nano-eGFP-mono. The negative control nano-HER2 was not detecting YFP-polη, as expected. All together these results demonstrate the superiority of the bivalent formats compared to the monovalent counterpart.

Moreover, the nano-GFP-E3 is in some cases better than the nano-eGFP-tandem, justifying the use of the small E3 peptide as dimerization device.

DISCUSSION

For particular applications, bivalency might be advantageous or even required. For example, only low-nanomolar or subnanomolar range antibodies or antibody fragments can be used as successful imaging agents (Anna [START_REF] Orlova | Affibody Molecules for Molecular Imaging and Therapy for Cancer[END_REF]. Thus, bivalency is a simple way to increase affinity by means of avidity. V H Hs are perfect building entities to achieve multivalent units. This can be achieved either by genetic fusion of V H H open reading frames in tandem, by using dimerization domains, or by chemical conjugation. However, the formatting of bivalent V H Hs is not strictly straightforward and each method has its advantages and drawbacks [START_REF] Conrath | Camel Single-domain Antibodies as Modular Building Units in Bispecific and Bivalent Antibody Constructs[END_REF].

Here, we report the use of a small peptide of 31 amino acid residues to engineer bivalent V H Hs. This peptide, E3, derives from the tetramerization domain of p53 and is capable to self-associate spontaneously into extremely stable homodimers. Indeed, the K D value of the E3 monomer-dimer equilibrium was evaluated to be lower than 10 -15 M [START_REF] Brokx | A Stable Human p53 Heterotetramer Based on Constructive Charge Interactions within the Tetramerization Domain[END_REF]. In comparison, the K D of a small leucine zipper dimerization domain was shown to be of 2.5 10 -7 M [START_REF] Moll | Designed heterodimerizing leucine zippers with a range of pIs and stabilities up to 10-15 M. protein science[END_REF]. In addition, we have previously shown by Circular

Dichroism that the unfolding state of the E3 construct after thermal denaturation was almost completely regenerated upon cooling, suggesting an important resilience of the secondary structures [START_REF] Vigneron | Self-Associating Peptides for Modular Bifunctional Conjugation of Tetramer Macromolecules in Living Cells[END_REF].

The fusion at the C-terminus of the anti-eGFP V H H leading to a tail-to-tail orientation was evaluated against a fusion of the V H H in tandem, with a tail-to-head orientation. If the ELISA and SPR results showed moderate gains of affinity, as compared to the monomeric parental V H H, this might be explained by the fact that the monomeric parental V H H used in this study has already an extremely high affinity towards the eGFP (K D of 0.32 nM [START_REF] Saerens | Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies[END_REF]). Nevertheless, differences could be observed in SPR between the tandem and the dimeric format in terms of the maximal value of the normalized response (R norm ). The R norm reached the maximal value of 1.00 for the nano-eGFP-E3, but only around 0.7 for the nano-eGFP-tandem. Thus, one can hypothesize that the second V H H in the tandem construct has a slightly decrease of affinity for the eGFP. Indeed, in some cases, it has been observed that in tandem constructs, the C-terminal V H H exhibited slightly reduced on-rate properties than the N-terminal moiety, leading to a reduced affinity [START_REF] Conrath | Camel Single-domain Antibodies as Modular Building Units in Bispecific and Bivalent Antibody Constructs[END_REF]. For example, two bivalent versions on a V H H directed against the scorpion toxin AahI have been compared [START_REF] Hmila | VHH, bivalent domains and chimeric Heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI′[END_REF], one construct correspond to a tandem V H H spaced by a 17 amino-acid peptide, and the other one correspond to the V H H fused to the human IgG1 CH2-CH3 domain which spontaneously dimerises. The homodimer construct displayed an apparent dissociation constant of around 50-fold better than the tandem V H H.

We also compared the binding activity of the three V H H constructs on various eGFPfusion proteins localized in different cell compartments. For classical immunofluorescence on H2B-eGFP cell line, both bivalent were better at detecting the eGFP antigen than the monovalent format. However, only the nano-GFP-E3 was applicable at a concentration below 1 nM. For soluble eGFP fusion proteins, like eGFP-β-galactosidase, there were no noticeable differences between the monovalent and both bivalent V H Hs. In contrary, the detection of PCNA through the binding of the V H Hs on the chimera fusion protein (P40M1-eGFP-con1)

clearly showed the superiority of the bivalent formats over the monovalent counterpart.

However, for soluble eGFP antigen such as MTS-eGFP directed to the mitochondrial compartment, the expression level of the nanobodies was to high to be titrated by the transiently expressed eGFP antigen. We could overcome this problem by electro-transferring the purified V H Hs into the living cells. Again, all three nanobodies co-localized with the mitochondrial-expressed eGFP, with a slight better efficiency for both bivalent formats.

When analyzing the chromatin-bound eGFP polymerase η fusion antigen, both bivalent formats were clearly betters compared to the monovalent counterpart. While 30% of nano-eGFP-mono co-localized with the antigen, more than 85% of the dimer E3 format was associated with the YFP-polη signal. One could notice that again, the nano-eGFP-E3 gave a clearer signal than the nano-eGFP-tandem.

CONCLUSION

This paper described a self-associating E3 peptide able to form spontaneously and remarkably stable homodimers in vitro and in living cells to engineer bivalent V H Hs leading to tail-to-tail format. Because V H Hs display unique properties, such as high solubility and a reversible unfolding reaction under stringent conditions [START_REF] Muyldermans | Nanobodies: natural single-domain antibodies[END_REF], E3 small peptide is of particular interest for dimerization of V H Hs. Indeed, we have previously reported that E3 peptide is robust at low pH, form in complex media and resists thermal denaturation above 90°C [START_REF] Stoessel | Modular Conjugation of a Potent Anti-HER2 Immunotoxin Using Coassociating Peptides[END_REF][START_REF] Vigneron | Self-Associating Peptides for Modular Bifunctional Conjugation of Tetramer Macromolecules in Living Cells[END_REF], making it a powerful tool for bivalent V H Hs engineering.

Supporting Information Available

DNA and proteins sequences, figures showing additional representations of the nanobodies, purification steps and SEC analysis of recombinants proteins, and immunofluorescence assays are given.

  sequences encoding for nano-eGFP were synthetized by Integrated were then inserted either into pETOM digested by NcoI-EcoRI or into the pET-con1-E3 digested with NcoI-SpeI described elsewhere[START_REF] Vigneron | Self-Associating Peptides for Modular Bifunctional Conjugation of Tetramer Macromolecules in Living Cells[END_REF] given rise to the pETOM-nano-eGFP-mono and pETOMnano-eGFP-E3 expression vectors, respectively. For the pETOM-nano-eGFP-tandem, an optimized sequence encoding the nano-eGFP followed by (G4S) 3 linker was synthetized cloned into the pETOM-nano-eGFP-mono digested by NcoI giving rise to pETOM-nano-eGFP-tandem.To generate eukaryotic expression vectors used in this study, the nano-eGFP fused or not to E3 sequence, and the nano-eGFP-tandem coding sequences were first amplified by PCR from pETOM-nano-eGFP-mono, pETOM-nano-eGFP-E3 and pETOM-nano-eGFPtandem vectors, respectively using the described primers. The fragments were digested with NcoI and SpeI restriction enzyme, and cloned into the pßA-based vector(Vigneron et al., was cloned in NdeI site of the pET-Histag-eGFP. Sequences of DNA constructs were verified by DNA sequencing. The β-Gal-eGFP, β-Gal-NLS-eGFP, P40M1-eGFP-con1 and YFP-DNA polymerase η expression plasmids have been described

Figure 1 :

 1 Figure 1: Schematic representations of the nano-eGFP-tandem, nano-eGFP-E3 and nano-eGFP-mono.

  -E3 displayed a single peak corresponding to a homodimer. The final yields of purified proteins were between 50 and 80 mg/L.

Figure 2 :

 2 Figure 2: Functional binding properties of the nanobodies. (A). Apparent binding constants of the nanobodies based on valence concentration were measured by indirect ELISA against immobilized purified eGFP at a concentration of 2 µg/ ml. Binding curve averages of three independent experiments are shown. Apparent binding constant is 4.67 ± 0.99 nM for the nano-GFP-mono, 0.44 ± 0.02 nM for the nano-GFP-E3 and 0.63 ± 0.04 nM for the nano-eGFP-tandem. (B) Kinetics analysis of the nanobody interaction followed by Surface Plasmon Resonance. Are shown the normalized signal over time after double referencing. The normalized signal takes into account the immobilized ligand level and the

  20 nM and 4nM, all three nano-GFP formats were detecting the endogenous nuclear H2B-

Figure 3 .

 3 Figure 3. Detection of the eGFP in fixed and permeabilized HeLa cells stably expressing H2B-eGFP fusion protein by conventional immunofluorescence. The binding of the purified nanobodies to H2B-eGFP was detected using the anti-Myc 9E10 monoclonal antibody (mAb) followed by goat anti-mouse Ab-conjugated with Alexa Fluor 568 (red). Scale bar 20 µm.

Figure 4 .

 4 Figure 4. Binding recognition of various eGFP fusion proteins by anti-eGFP nanobodies A and B U2OS cells were transfected with various eGFP antigen expression vectors as indicated, together with nanobody expression plasmids. At 24 h post-transfection, cells were treated (P40M1-eGFP-con1) or not with CSK buffer (eGFP-β-Gal antigens), fixed, permeabilized and mounted. The proteins fused to eGFP were visualized by direct fluorescence and bound-nanobodies were detected by the anti-Myc 9E10 monoclonal antibody (mAb) followed by a labeled Alexa 568 anti-mouse. Scale bar, 10 µm.

  left panel). There were no nano

Figure 5 .

 5 Figure 5. The purified nanobodies were directly electro-transfected in (A) H2B-eGFP cell line or together with the MTS-eGFP expression plasmid, and (B) in MRC5 stable cell line expressing YFP-polη. At 24 h post-transfection (B) cells were treated with UV radiations and treated or not with CSK buffer, as indicated. Cells were fixed, permeabilized and mounted. The eGFP antigens were visualized by direct fluorescence (green) and nanobodies were detected by the anti-Myc 9E10 monoclonal antibody (mAb) followed by a labeled Alexa 568 anti-mouse (red). Nuclei were visualized by DAPI (blue). Images were taken by fluorescence microscopy and phase-contrast (PC). Scale bars, (A) 20 µm, and (B) 10 µm. (C) Statistic analysis were performed on MRC5 stable cell line expressing YFP-polη transduced with the nanobodies, as indicated, and after CSK treatment before cell fixation and permeabilization.

ACKNOWLEDGEMENTS

This work was supported by funds and/or fellowships from the Centre National de la Recherche Scientifique, the University of Strasbourg, the French Ministry of Research and the Ligue Régionale contre le Cancer (CCIR-GE), the Fondation ARC pour la recherche sur le cancer et ITI Innovec (IdEx (ANR-10-IDEX-0002) and SFRI (ANR-20-SFRI-0012).