Jean-Marc Dubost

Vichith Lamxay

Sabrina Krief

Michael Falshaw

Chanthanom Manithip

Eric Deharo
email: eric.deharo@ird.fr

From plant selection by elephants to human and veterinary pharmacopeia of mahouts in Laos

Abstract

Ethnopharmacological relevance

To what extent has animal observation contributed to the development of human pharmacopeias? We approach this question here through the study of mahouts' knowledge regarding the responses by elephants to their health problems, and the human medicinal uses of plants and the care of domestic animals that result from their observations. Materials and methods 32 mahouts were interviewed in Thongmyxay district and 28 at the Elephant Conservation Center in the province of Xayabury. Interviews focused on the elephants' diet, health problems, plant items they consume in particular physiological or pathological contexts and the treatments that mahouts provide them. For each plant mentioned, the part of the plant consumed and mode of preparation and administration if used by mahouts were recorded. Species samples were then collected and later identified by specialists. Results 114 species were recorded as being consumed by elephants during interviews with mahouts and forest outings with them to collect samples. Twenty species were identified as used by elephants in particular pathological conditions or physiological states. According to interviewed mahouts, the consumption of certain plants improves the health of the elephant. We observed clear convergences between the observations interpreted by the mahouts as selfmedication behaviour from elephants and their own medicinal practices (for human and veterinary purposes).

Conclusion

Beyond a mere reproduction of elephant self-medication behaviours observed, the human or veterinary medicinal cares derived from these observations are the result of complex arrangements integrating all available medicinal and conceptual resources into elaborate preparations. We recommend that mahouts' knowledge about traditional medicinal care given to elephants be further compiled, as it could have a beneficial impact on veterinary health care provided in elephant resorts and elephants' well-being.

Introduction

The study of animal self-medication behaviour and zoopharmacognosy (Rodriguez et Wanghram, 1993) began about 20 years ago with scientific observations made about primates, especially chimpanzees [START_REF] Wrangham | Relationship of chimpanzee leaf-swallowing to a tapeworm infection[END_REF][START_REF] Huffman | Seasonal trends in intestinal nematode infection and medicinal plant use among chimpanzees in the Mahale Mountains, Tanzania[END_REF]. However, this type of behaviour can be found in many accounts from traditional oral cultures that attribute an animal origin to certain medicinal uses of plants [START_REF] Huffman | Animal self-medication and ethno-medicine: exploration and exploitation of the medicinal properties of plants[END_REF] as well as in ancient literature (Boeme, 2008). Studies have documented convergences of therapeutic uses of some plants between animals and humans [START_REF] Huffman | Self-Medicative Behavior in the African Great Apes: An Evolutionary Perspective into the Origins of Human Traditional Medicine[END_REF][START_REF] Krief | Ethnomedicinal and bioactive properties of plants ingested by wild chimpanzees in Uganda[END_REF][START_REF] Krief | Diversity of items of low nutritional value ingested by chimpanzees from Kanyawara, Kibale National Park, Uganda: an example of the etho-ethnology of chimpanzees[END_REF][START_REF] Morrogh-Bernard | Self-medication by orang-utans ( Pongo pygmaeus ) using bioactive properties of Dracaena cantleyi[END_REF] suggesting either independent convergences, or transfers of knowledge from animals to humans. A few cases of transfers have been reported where direct observation of a sick animal's behaviour can be linked to the subsequent and effective use of the same remedy by humans presenting the same condition [START_REF] Huffman | Self-Medicative Behavior in the African Great Apes: An Evolutionary Perspective into the Origins of Human Traditional Medicine[END_REF][START_REF] Krief | L'interspécificité du pharmakôn dans le parc Kibale (Ouganda) : savoirs partagés entre humains et chimpanzés ?[END_REF]. However, there has been little exploration of how medicinal practices are adapted and shaped by the observation of animal behaviour. During a survey on traditional plant uses conducted in southern Thailand in 2014, we looked at the knowledge of two mahouts (the English term borrowed from India for a person in charge of a domestic elephant in Asia) that suggested that this particular human-animal relationship between mahouts and their elephants can be a significant source of knowledge to better understand the origin of human medicine [START_REF] Jean-Marc | Medicinal knowledge input from animal observation: the case of Asian elephant feeding observations linked to specific medicinal uses in Chumphon province -Thailand[END_REF]. Indeed, in the traditional way of managing elephants within the village context, elephants are not fed but are allowed to feed themselves in the surrounding forests [START_REF] Maurer | Wild-captive interactions and economics drive dynamics of Asian elephants in Laos[END_REF][START_REF] Maurer | Conservation de l'éléphant d'Asie (Elephas maximus) par l'étude des interactions entre humains et populations sauvages et semi-captives d'éléphants: une approche intégrée des dimensions démographiques, génétiques[END_REF]. Because of the comparable life span between elephants and humans, mahouts who spend a large part of their lives with the same animal are able to observe its needs and responses according to its state of health, responses that they sometimes interpret as a form of self-medication from these animals. Moreover, the traditional care they provide elephants places mahouts at the interface of these presumed animal self-medication behaviours and local traditional medicine, providing a good opportunity to study the convergence of medicinal plant use between men and elephants. We therefore carried out a study to explore the relationship between traditional human and veterinary medicine and observations on elephant behaviour by mahouts in an area of Laos where mahouts and owners still keep their elephants in the vicinity of villages. Data obtained were supplemented with elephant observations and interviews conducted in an elephant center dedicated to the conservation of this species in Laos.

Background

Laos is located in a humid tropical zone. The climate is seasonal, with a rainy season (May-October) a cool-dry season (November-February) and a hot-dry season (March-May). Characterized by extensive forest cover, the country is part of the Indo-Burma biodiversity hotspot [START_REF] Myers | Biodiversity hotspots for conservation priorities[END_REF], but this cover has drastically decreased and is becoming more and more fragmented. In the mid-1960s, forested areas accounted for 70% of the territory, compared to 41% in 1999 [START_REF] Southavilay | Timber trade and wood flow study for Lao PDR 30[END_REF] and areas of over 1000 ha decreased from 88% of the total forest area in 1992 to 54% in 2004 [START_REF] Evrard | La mise en oeuvre de la réforme foncière au Laos : impacts sociaux et effets sur les conditions de vie en milieu rural, LSP Working Paper[END_REF], indicating a process of landscape fragmentation. However according to the most recent assessment of forest cover by the Department of Forestry of the Lao PDR, reduction of forest cover decreased significantly from 2002 to 2010, and according to official sources forest cover has increased slightly in the north of the country [START_REF] Kim | Forest policy measures influence on the increase of forest cover in northern Laos[END_REF].

Elephas maximus in Asia and Laos

The Asian elephant Elephas maximus is classified as an endangered species by the IUCN. The loss and fragmentation of its habitats is the main cause of this decline of elephant populations in Asia (Leingruber et al., 2003), where it is found in 13 countries (IUCN, 2016). In Laos, the elephant is an emblematic species, and the country was previously called Lan Xang "Land of the million elephants". However, the population estimated at [START_REF] Myers | Biodiversity hotspots for conservation priorities[END_REF]-3000 wild elephants in 1988(Phantavong and Santiapillai, 1992) had reduced to just 600-800 individuals by 2009(Khoumboline, 2011)). Domestic elephant numbers are also declining sharply in Laos and are now comparable to the number of their wild counterparts [START_REF] Khounboline | Current status of Asian elephants in Lao PDR[END_REF][START_REF] Suter | Population viability of captive Asian elephants in the Lao PDR[END_REF]. These elephants living in contact with humans have an intermediate status between wild and captive or domesticated animals. Traditionally sourced from wild populations [START_REF] Lair | Gone astray. The care and management of the Asian elephant in domesticity[END_REF][START_REF] Sukumar | The living elephants: evolutionary ecology, behavior, and conservation[END_REF], they have not undergone a selection process like most domesticated animals. According to traditional management methods, they are not fed and are periodically released in the forest over long periods [START_REF] Maurer | Conservation de l'éléphant d'Asie (Elephas maximus) par l'étude des interactions entre humains et populations sauvages et semi-captives d'éléphants: une approche intégrée des dimensions démographiques, génétiques[END_REF]. It is therefore equally inappropriate to apply the epithet of captives to them, which refers instead to animals whose movements are much more restricted. We will thus use for the elephants of Thongmyxay district the term "village elephant" proposed by Maurer (ibid) and used by [START_REF] Lainé | Travail interespèces et conservation[END_REF] and will use "captive elephant" when talking about elephants kept in camps. Village elephants often have several owners within the same family, but usually just one will be in charge of looking after them and is described as their mahout [START_REF] Suter | Changes in Elephant Ownership and Employment in the Lao PDR: Implications for the Elephant-Based Logging and Tourism Industries[END_REF].

Diet and Habitat

Elephants are generalist postgastric herbivores that feed on a wide variety of plant items, whether in terms of species consumed, life form (trees, vines, grasses, herbs) or of parts selected such as leaves, fruits, twigs, barks, roots, woody stems [START_REF] Sukumar | The Asian Elephant: Ecology and Management[END_REF][START_REF] Sukumar | The living elephants: evolutionary ecology, behavior, and conservation[END_REF][START_REF] Chen | Diet composition and foraging ecology of Asian elephants in Shangyong, Xishuangbanna, China[END_REF][START_REF] Himmelsbach | Food plants of captive elephants in the Okkan Reserved Forest, Myanmar (Burma), Southeast Asia[END_REF][START_REF] Joshi | Feeding behaviour of wild Asian elephants (Elephas maximus) in the Rajaji national park[END_REF]Campz-Arceiz et al., 2008;[START_REF] Santra | Nutritional status of forage plants and their use by wild elephants in South West Bengal, India[END_REF]Lithong et al., 2007;[START_REF] English | Plant selection and avoidance by the Bornean elephant (Elephas maximus borneensis) in tropical forest: does plant recovery rate after herbivory influence food choices?[END_REF]. Areas which constitute ecotones between cultivated areas and regrowth or disturbed forests colonized by pioneer species, which display higher levels of understory vegetation than old-growth forest, are optimal for elephants in terms of resources and carrying capacity [START_REF] Fernando | Range-wide status of Asian elephants[END_REF][START_REF] Fernando | Perceptions and Patterns of Human-elephant Conflict in Old and New Settlements in Sri Lanka: Insights for Mitigation and Management[END_REF][START_REF] Evans | Underproductive agriculture aids connectivity in tropical forests[END_REF]. These characteristics regularly bring elephants to frequent the outskirts of anthropized areas.

Material and method

Study area

This study was conducted in Xayabury province (Figure 1), which is home to 80% of the village and captive elephants in Laos [START_REF] Lassausaie | Tuberculosis in Laos, who is at risk: the mahouts or their elephants?[END_REF], in two sites: 1. In the district of Thongmyxay, whose inhabited area is encompassed by the Namphuy National Protected Area (NNPA) which covers 1912 km² (WWF, 2019) and shelters the second largest population of wild elephants in Laos, estimated at 60-80 individuals in 2009 (Khoumboline, 2011). The inhabited area of Thongmyxay is mainly surrounded by forested areas consisting of degraded secondary forests, Dypterocarpaceae forests, riverine forests and hill bamboo forests. 2. At the Elephant Conservation Center (ECC) in the north of the province. This center, dedicated to wild and captive elephant conservation and research in Laos, develops ecotourism activities focused on elephant observation. It is delegated by the Ministry of Forests to be responsible for the NNPA's wild elephant protection activities, and has a concession of 512 ha where elephants feed themselves. Vegetation consists mainly of young, dry dipterocarp forest dominated by Shorea siamensis Miq. within a broad topographic range covering the area and an abundance of bamboo in patches running along the bank of a stream. Signs of logging are present and the area is surrounded by rice farms.

Survey

We carried out an inventory of plants which the mahouts indicated were consumed by elephants, specifying the parts consumed. Then we organized forest outings with mahouts (in Thongmyxay district and at ECC) to explore different areas where they let their elephants feed, in order to collect samples of the listed plants, which were dried to prepare herbarium specimens. The taxonomic identification was carried out under the responsibility of Vichith Lamxay in the Department of Botany at the National University of Laos. Plants names were checked and updated according to the standard database "The Plant List" (http://www.theplantlist.org). Voucher specimens were deposited at the Herbarium of the National University of Laos in Vientiane. We conducted semi-structured interviews with 60 mahouts -32 in Thongmyxay district (aged 38 to 78) and 28 in ECC (aged 35 to 63) -all of whom were Tai-Lao, the dominant ethnic group in Laos. The interviews focused on:

-Elephant management practices and changes that have occurred since the beginning of their mahout activity, especially issues of access to food resources, in order to assess the context of the observations reported by mahouts. -The feeding behaviour of their elephants, by requesting qualitative information on the context of consumption of the plant items they consume. -Elephant health problems, their needs, the possible care provided (including preparation and administration methods) and observations regarding how elephants respond to their own problems. When plants were cited as being consumed by elephants in a particular physiological context, mahouts were also asked if they used them themselves in the family environment.

We also followed a female elephant that had been released into the forest (on average six hours a day for five days) at the ECC site to observe its feeding behaviour. This enabled us to get a better understanding of the observations reported by the mahouts and thus inform our discussions with them on the behaviour of their elephants.

Ethics and authorizations

Ethical approval was obtained from the National Ethics Committee for Health Research (NECHR) under the supervision of the Ministry of Health of the Lao PDR (0894NIOPH/ NECHR). Authorization to collect the plants was obtained from the relevant authorities. The study was approved by the local Nagoya protocol office (N°008/SABS).

Voucher specimens were deposited at the herbarium of the Department of Biology, Faculty of Sciences, National University of Laos (NUoL), in Vientiane.

Results and discussion

Elephant management practices in Thongmyxay district

In recent decades, there have been three successive phases in the evolution of elephant management and use. Of the 32 mahouts interviewed in Thongmyxay district, 21 are no longer active, either because they have sold or rented out their elephant (cf. infra) or because they have handed over their responsibility to relatives. They described the traditional elephant management system that prevailed before the construction of the road linking Thongmyxay district, which was built 20 years ago. Elephants were then used mainly for the transportation of goods -mostly rice after the November harvests -and on demand for occasional villagescale logging to provide locally-used timber. These activities took place in the dry season from November to April when the land is passable. During this period, elephants were tethered (with a 30-40 m chain) or simply hobbled in the wooded areas near the village, and were moved once or twice a day to renew their feeding areas and to allow them to drink.

During the rainy season, when all available human resources were used to plant crops, elephants were released in small groups in the forest that is now part of the NNPA. They were visited from time to time and sometimes came back on their own at the beginning of the dry season, or the mahouts would go looking for them and bring them back. About thirty years ago, with the development of the road network and the arrival of vehicles, elephants were no longer used for transport. They were used instead for logging, meeting the demand of forestry companies who were then engaged in a massive exploitation of timber wood. [START_REF] Labatut | The Veterinary Care of Domesticated Elephants in Laos by a Mobile Veterinary Unit[END_REF]. As a result, since elephants no longer have any use in terms of work except for occasional logging for the villagers, the owners have tended to free themselves from the burden that their animal now represents by selling it (11 respondents) or renting it out to elephant camps (8 resp.). However, other owners in Thongmyxay chose to keep their elephants (11 resp.). This is due firstly to the fact that the district enclosed in the NNPA offers crop-free areas where they can still release their elephants. Secondly, this protected area, which houses the second largest population of wild elephants in Laos, provides opportunities for the new income activity of elephant breeding for sale. The female elephants are thus left in the forest to mate with wild males (reported by 6 mahouts out of the 11 still working) and an elephant calf can be advantageously sold or rented to elephant camps for which it is a firstchoice attraction [START_REF] Maurer | Wild-captive interactions and economics drive dynamics of Asian elephants in Laos[END_REF].

Inventory of plants indicated by mahouts as being consumed by elephants

When they keep their elephant tethered, mahouts can see which species have been consumed by observing changes in the vegetation cover of the area where their elephant has been feeding and the marks left on specimens. The daily movements to bring the elephant to a new feeding area or the regular visits to them when released also provide an opportunity for many direct observations of plant consumption. A total of 114 species were recorded as being consumed by elephants during mahout interviews and forest outings with them, with the parts taken for each amounting to 197 plant items. However, according to all mahouts, bamboo twigs and leaves make up the staple part of the elephants' diet. This inventory will be the subject of a future publication. Parts of plants taken may include roots, stems, twigs, bark, wood, leaves, fruits, and young shoots. Plant forms are also very diverse, ranging from mature trees, saplings and vines to grasses. Other than the consumption of plants, 19 mahouts report the occasional geophagous behaviour of elephants, this issue will be discussed in section 4.4.

Observing elephant feeding behaviour has demonstrated the high selectivity that an individual can show in the choice of items consumed, uprooting a tree to consume only the root (Figure 2), or stripping twigs to keep either the stem or the leaves. The elephant often first brushes the plant item with the tip of its trunk before choosing to consume it or not, and uses a whole range of techniques involving the trunk, front legs, tusks and jaws, and the animal's weight and strength. Some parts such as tree roots and bark require a sustained effort to be collected and are consumed in significant quantities (for example, a trunk 15 cm in diameter can be debarked by 50 cm in about 20 minutes).

Knowledge about elephant health

The elephant is perceived by mahouts as an animal with generally few health problems, and they explain that in most cases the elephants know how to heal themselves using the resources around them. When asked what they need to do to keep an elephant healthy, mahouts say that the most important thing is to make sure that it can find what it needs on its own. Elephants should therefore be released as often as possible (5 resp.). When tethered, suitable places with abundant and diversified vegetation should be carefully chosen (15 resp.) and the elephant moved once or twice a day to allow it to drink and renew its feeding area. In these conditions, mahouts say that elephants rarely experience health problems. They evaluate the condition of their elephant using various signs. When it is tethered and they go to fetch it, the faeces that are present inform them of its digestive state: diarrhoea, constipation (hard or absent), or presence of macroscopic parasites. A look at the browsed area tells them if the elephant has a normal appetite and what has mainly been eaten. A pathological condition is often reflected in the fact that the elephant sleeps a lot during the day, seems apathetic and eats little (7 resp.), sometimes by an abnormal dryness of the interdigital space which is normally wet because here elephants have glands similar to human eccrine sweat glands (Fowler and Mikota, 2008).

Among the health problems mentioned that could affect elephants are firstly digestive disorders (35 resp.) characterized by diarrhoea (31 resp.), constipation (4 resp.) or bloating (3 resp.). Also mentioned are accidental injuries or abrasions due to the chain used to tether elephants (11 resp.), purulent abscesses (9 resp.), skin rashes (8 resp.), eye pain with discharge (5 resp.), and amidal (3 resp.) a local term that refers according to veterinarians to an infection by EEHV (Elephant Endotheliotropic HerpesVirus), and can be fatal to the calves that contract it [START_REF] Long | Review of Elephant Endotheliotropic Herpesviruses and Acute Hemorrhagic Disease[END_REF][START_REF] Bouchard | First reported case of elephant endotheliotropic herpes virus infection in laos[END_REF]. The term amidal is borrowed from the French word "amygdale" meaning "tonsil" because one of the visible symptoms of this condition in elephant calves, according to mahouts, is swelling of the throat area.

In a study conducted in South India [START_REF] Jayakumar | Ethno-veterinary practices in Southern India for captive Asian elephant ailments[END_REF] investigating ethnoveterinary care given by mahouts to their elephants, digestive disorders are also cited as the most frequent problems among the captive elephants considered. However, constipation is reported to be much more frequent than diarrhoea, unlike in our study where diarrhoea is the main elephant digestive disorder reported. The South Indian study also notes that intestinal disorders, among other problems observed, are related in particular to inadequate husbandry practices such as poor hygiene in the elephant housing area, lack of exercise and monotonous food. These conditions differ in many respects from those experienced by elephants in Thongmyxay District or the ECC, where:

-they probably access a much more diverse diet as they feed themselves in a forested area -they might spend much more time on feeding than fed animals -they stay permanently in natural areas and probably have more opportunities for exercise (even when tethered they are moved daily from one area to another and still have to obtain their own food). Such differences in elephant management practices may explain the different prevalence of constipation versus diarrhoea in the elephants in the South Indian study and ours.

In a report published about veterinary care provided to elephants by Elephant Asia (an NGO which had set up a mobile elephant care unit in Laos), abscesses, intestinal disorders, nails problems, eye problems and injuries were by decreasing order the main problems found in the 315 elephants examined [START_REF] Labatut | The Veterinary Care of Domesticated Elephants in Laos by a Mobile Veterinary Unit[END_REF]. Abscesses are most common among the ailments reported, which may be due to the fact that 78% of the elephants examined were working in the logging industry, where, according to the authors, abscesses were caused by the prolonged chaffing of chains.

When asked if there are periods when elephants are more vulnerable, out of the 13 mahouts who had an opinion, 9 mentioned the end periods and transitions of seasons (from dry to rainy season and vice versa) that they believe correspond to a change in food, indicating for example that tender grasses enjoyed by elephants grow back at the beginning of the rainy season. [START_REF] Sukumar | The living elephants: evolutionary ecology, behavior, and conservation[END_REF] also notes the appearance of intestinal disorders at the changes of seasons and hypothesizes that they can be induced by a change of diet (pre-eminence of browsing in the dry season and grazing in the rainy season) modifying the fiber/protein ratio of their food, to which their digestive system must readapt. Conversely, according to some mahouts, at the end of the dry season the decline in the quality of the available water, which is more stagnant, may also be the cause of these disorders.

Elephant behaviour interpreted as self-medication

-Soil use: When an elephant has localized pain or discomfort (bite, sting, or abscess), it tends to blow on it (4 resp.), drawing attention to a possible external problem on a less visible area of its body, or may also apply mud on the affected area (7 resp.). While mahouts observe regular geophagic behaviour, three of them report that they have observed this type of behaviour particularly when their animal has intestinal problems, with a subsequent improvement noted. These geophagous behaviours are documented in the literature and the composition of soil licks frequented by African (Loxodonta cyclotis and africana) and Asian (Elephas maximus) elephants has been analyzed in the Central African Republic [START_REF] Klaus | Geophagy by large mammals at natural licks in the rain forest of the Dzanga National Park, Central African Republic[END_REF], Zimbabwe [START_REF] Holdø | Geophagy in the African Elephant in Relation to Availability of Dietary Sodium[END_REF] and Sri Lanka [START_REF] Chandrajith | Geochemical and mineralogical characteristics of elephant geophagic soils in Udawalawe National Park, Sri Lanka[END_REF]. Klaus and Holdø note a higher mineral richness in the soils that could compensate for deficiencies in elephant feeding, but Chandrajith does not note significant differences in mineral elements between soil licks and surrounding soils. Klaus and Chandrajit suggest that the clay elements of these soils could help detoxify some of the elements ingested by elephants by adsorbing secondary toxic compounds.

-Plant use: In addition to blowing or applying mud to an affected area and geophagy in the event of intestinal problems, of the 49 mahouts interviewed with at least 10 years' experience, 26 reported observations of search behaviour by their elephants and increased consumption of specific items when they have health problems or in relation to their physiological state. We will call this search behaviour and increased consumption of specific plant items described by mahouts "privileged consumption". For the same condition or physiological state of the elephant, mahouts report a privileged consumption of a single plant item (6 resp.), or of several species, with overlaps that vary from one to the other (19 resp.). We will name this here Elephant Use Report (EUR) referring to an observation described as recurrent by one mahout, where a particular pathological condition or physiological state identified in the elephant they care for is associated with the search behaviour and consumption of one or more items from the same plant species.

We have thus identified 64 EUR in which 20 species are mentioned, representing 30 plant items. These EURs presented in Table 1 fall into two main categories, one relating to health disorders and the other related to female reproduction including gestation, ante and postpartum, and lactation periods. For convenience, we have grouped this last category of EURs, under the generic term of "motherhood". This group of EURs is detailed in Table 2. Diarrhoea is the main context of these plant item intakes: 43 EUR, indicated by 19 mahouts with 19 species collected representing 25 items; this is also the elephant health problem most often mentioned by mahouts. Then comes motherhood: 18 EUR indicated by 8 mahouts with 7 species mentioned and 8 items consumed. Other contexts were mentioned by only one mahout for each: dizziness (the elephant shakes), weakness, and 'fever', which is described as an apathetic state with loss of appetite.

The 30 items mentioned in these EURs are included among the 197 items recorded in our inventory of items consumed by elephants. When the mahouts specified the reasons for the self-medication interpretation they give to the recorded EURs, two criteria emerged:

-Firstly, the mode of consumption is in relation to the reported condition. Privileged consumption is the general case, in which the item is part of the elephant's usual diet but is particularly sought after and consumed in much greater quantities in certain contexts. Alternatively, exclusive consumption is where the item is only consumed in a particular context (19 EUR -11 resp.). It should be noted that according to each mahout's own observations, for the same EUR some mahouts mention an exclusive mode of consumption of the plant item for a specific ailment, while others may report the consumption of the same item as part of the elephant's usual diet.

-Secondly, the consumption of the item is followed by a rapid improvement in the condition of the elephant.

Beyond the observation of the consumption of specific items in a particular physiological context, additional information and observations were provided by mahouts around the question of the interpretation of self-medication by elephants. Five of them have observed that elephants consume certain roots when they have diarrhoea (Harrisonia perforata Blanco Merr. for two of them, Castanopsis indica Roxb. ex Lindl. A.DC. for another and Terminalia mucronata Craib & Hutch. for the last) and have noted that it improves their condition. Knowing this, if their animal suffers from diarrhoea, they will bring it to a place where these plant resources are available. They say that when they return to move the elephant, these plants have largely or entirely been uprooted, their roots consumed, and that the elephant's condition is improving rapidly. The efficacy of these practices to improve the condition of their elephants has not been quantified or demonstrated, but this approach has in a way acted as a test and has confirmed for the mahouts their interpretations of elephant self-medication based on the plant selection behaviour of the sick elephants they observe.

Interactions between elephant observations and local medicinal practices

Going further than just facilitating access to plant resources, some mahouts also provide them to their animal. Four mahouts report that when their elephant suffers from diarrhoea, they collect and bring it the roots that they know it seeks when it experiences this condition. These are H. perforata (2 resp.), T. mucronata (1 resp.), Spondias pinnata L.f. Kurz (1 resp.). They sometime add salt to make the items more appetizing but the items are given in their raw form. In another similar case though it leads to a more elaborate practice, where the mahout had noted that when the elephant has what he calls a fever (which to him means his elephant is sad and listless) it consumes much more of the Tinospora crispa L. Hook.f. & Thomson vine and recovers quickly. If he observes this condition in his animal, he crushes the root and stem of this vine with salt and gives it to be eaten. When he passes through a place where this vine is abundant, the mahout collects some of it and dries it to have some at hand when needed.

These are examples of local veterinary care practices resulting from the observation of elephant behaviour. In the last case described this leads to the creation of a reserve of a therapeutic resource, in a more elaborate form suitable for its conservation that can quickly be used in an assimilable galenic preparation (the root is dried and then reduced to powder with salt added before administration). We see here a veterinary preparation involving the observation of specific elephant behaviour (for the choice of ingredients) and an element from the mahout's medicinal background (in the galenic form). But this integration of elements derived from elephant observation and from the mahout's medicinal background into preparations given to elephants does not only concern the ingredients used and their processing. The medicinal background of mahouts may also be involved in the explanations that sometimes come with the interpretation of self-medication given to some of the elephants' behaviour. These explanations can in turn lead to the addition of other ingredients in preparations given to cure elephants. Thus two mahouts explain the consumption of certain plants by elephants by referring to their warm or cold qualities according to a classification of diseases and remedies in use in Laos for human medicine [START_REF] Souk-Aloun | La médecine du bouddhisme Théravâda au Laos : avec index des plantes médicinales indochinoises[END_REF][START_REF] Pottier | Le végétal dans la pharmacopée traditionnelle lao[END_REF][START_REF] Pottier | Yû dî mî hèng, être bien avoir de la force : essai sur les pratiques thérapeutiques lao[END_REF]. One mahout states that diarrhoea is a disorder of the category ok hon (literally 'feel hot') that the elephant treats by eating ya yen plants (literally 'remedy cool') such as H. perforata or Lithocarpus auriculatus (Hickel & A.Camus) Barnett. Two other mahouts report that when elephants suffer from tremors (1 resp.) or diarrhoea (1 resp.), they consume bitter roots. In the same vein, another mahout told us about this use that in his conception, diarrhoea results from excessive consumption of sour substances such as tamarind fruit, a disorder that he believes can be treated by consuming bitter substances. He noticed that when his elephant has diarrhoea it consumes more T. mucronata bark than usual. He himself uses this bark with that of H. perforata against diarrhoea, as it is a family remedy. Therefore, when his elephant is affected by diarrhoea and is tethered, he brings it bark of H. perforata and T. mucronata and adds bitter edible leaves -steamed and then salted -to strengthen the effectiveness of the remedy. This notion of sour/bitter balance is one of the components of a correspondence system found in traditional Lao medicine. In this system the properties of remedies are ordered by associating flavours with the hot/cold polarity mentioned above: sour and salty flavours being "spicy" are hot, while bitter and sweet flavours being "bland" are cold. Hence the possibility of correcting a "hot" imbalance due to an excess of sourness through the consumption of bitter substances of a "cold" nature [START_REF] Pottier | Yû dî mî hèng, être bien avoir de la force : essai sur les pratiques thérapeutiques lao[END_REF]). Through this last example, we have thus a veterinary preparation combining elements resulting from the observation of an animal's self-medication behaviour -or interpreted as such -(increased consumption of T. mucronata in case of diarrhoea), with elements borrowed from partially converging family uses (use of H. Perforata and T. mucronata in case of diarrhoea) and elements corresponding to concepts stemming from Lao traditional medicine (adding edible leaves for their bitterness). But this type of complex medicinal construction is not limited to veterinary uses: in Laos, it is common practice to macerate various vegetable or animal substances known for their therapeutic properties in alcohol, to be used for various ailments [START_REF] Vidal | La thérapeutique par les plantes au Laos[END_REF], but often taken as a fortifier [START_REF] Strigler | L'alimentation des Laotiens: cuisine, recettes et traditions au Laos et en France[END_REF]. One of the mahouts told us that when he sees his elephant eating roots, he takes some of them and puts them in alcohol to macerate, a preparation that he then consumes as a tonic. We have here an example of a human health practice that integrates data and knowledge built on the observation of the behaviour of animals frequented on a daily basis (elephants know how to treat themselves, they eat roots for that purposecf infra) into an already-existing traditional human practice (alcohol maceration of medicinal items).

Ethnoveterinary preparations

Table 3 shows ethnoveterinary treatments for elephants collected from 11 mahouts. Besides plant items, the ingredients used include clay, elephants' faeces and urine, and human urine.

Five out of the twelve species used in these treatments are listed in the EUR: Tinopora crispa, Entada rheedii Spreng., Spondias pinnata, Lithocarpus auriculatus and Harrisonia perforata.

Six species are used by several mahouts, but in different preparations given for different ailments: Alpinia galanga (L.). (2 resp.), Lasia spinosa (L.) Thwaites, (3 resp.), Pterocarpus macrocarpus Kurz (2 resp.), Spondias pinnata (L. f.) Kurz (2 resp.), Tinospora crispa) (2 resp), Harrisonia perforata (2 rep.). One of the mahouts uses the stem of the sappy Entada rheedii Spreng. vine on wounds and skin outbreaks. For the latter use, he soaks and twists it in water with detergent added. At first sight that may seem surprising, but the alkaline medium thus formed might facilitate the extraction of secondary active compounds from this vine. None of the plants used in these preparations are found in the above-mentioned study on ethnoveterinary care for elephants in South India [START_REF] Jayakumar | Ethno-veterinary practices in Southern India for captive Asian elephant ailments[END_REF] in which the authors trace the origin of ethnoveterinary care in India in scholarly traditions, particularly Ayurveda, recorded in treatises dating back to the earliest antiquity. This background may have diffused over time in the practices of mahouts in the region studied, but the possible continuities between these scholarly treatises and local mahouts' care for elephants using "indigenous plant-based medicines" are not considered as it is not the focus of this study. To our knowledge, there are some manuscripts in Laos dealing with elephant disease and care. In his study, [START_REF] Lainé | Travail interespèces et conservation[END_REF] mentions also manuscripts written in Tai or Pali which are used by traditional healers, the mo ya, but none of the mahouts we have interviewed mentioned or referred to such documents. The present study shows that the medicine provided to elephants rather reflects the individual practices and observations of each of the mahouts interviewed as illustrated by the examples given in Section 4.5. as well as by the fact that there is little overlap between the various preparations presented in Table 3. Fourteen mahouts use conventional veterinary medicine (three among these also use traditional treatments) to treat their elephants, mainly using antibiotic-based treatments for diarrhoea, abscesses and injuries. Four of them give or used to give their elephants energizers or vitamin supplements purchased from the veterinary pharmacy when their elephant is tired or to keep it in good shape. They use products sold for buffaloes and adjust the dose to be injected to their elephant.

Convergence between EUR and human medicinal uses

Six mahouts told us that sick elephants generally consume more roots than usual. Indeed, roots are present in 90% (58/64) of EUR. Interestingly, the preponderance of roots in Lao pharmacopeia was also highlighted in two studies conducted in Laos [START_REF] Delang | The role of medicinal plants in the provision of health care in Lao PDR[END_REF][START_REF] Libman | Medicinal plants: An important asset to health care in a region of Central Laos[END_REF]. Out of the 20 species mentioned in the EUR, 15 are also used by mahouts for therapeutic purposes in their households, and for 13 of these, the same part is used for the same type of indication (gastrointestinal disorder in all the cases mentioned). These convergences of medicinal uses found in the mahouts' own practices for treating human diseases and in the EURs could reflect interspecific convergences of medicinal uses of plants between humans and elephants. But it is also possible that the interpretation of selfmedication given to the consumption of medicinal items by elephants is induced by the mahouts' own medicinal uses of these plants. However, if we consider the uses of the H. perforata root, which is the subject of a significant number of EUR concerning diarrhoea (13 reports), seven of the mahouts told us that they use this item for the same indication in the household context while six did not know about any human use (Table 1). This rules out for the latter the possibility of induction of their own practices and knowledge in their selfmedication interpretation by elephants when they search for this item. In addition, many EUR do not have a counterpart in terms of medicinal uses known by mahouts for humans. This is the case in particular for the 18 EUR linked to the motherhood period in elephant females where the seven species mentioned are not known to them to be used by women for similar indications. This in itself is not surprising as among Tai-Lao people the medicinal knowledge about motherhood is mostly transmitted between women [START_REF] Pottier | Yû dî mî hèng, être bien avoir de la force : essai sur les pratiques thérapeutiques lao[END_REF] while the management of elephants is a male prerogative. The interpretation of self-medication linked to these EURs in this cultural context of sexual segregation of certain knowledge and activities appears also to be independent of the mahouts' medicinal knowledge. These aspects suggest that even if the medicinal background of mahouts may contribute a posteriori to account for certain behaviour observed in elephants (as for the hot or cold plants usescf. supra), it does not play a decisive role in the interpretation of self-medication that is given to them. It is more likely that the multiple convergences between the observations of privileged consumption of specific plant items by sick elephants and the use made by mahouts of the same items for the same type of pathology reflect convergences of medicinal plant uses between elephants and mahouts. These interspecific convergences could result either from independent acquisitions of uses in both species or from transfers of knowledge from elephants to humans. The closeness of life that is established in a daily relationship between mahouts and their elephants, and the few examples we have collected of the integration of behaviour observed in elephants into local veterinary and medicinal practices, such as the mahout collecting the roots consumed by elephants to prepare a medicinal maceration, rather pleads in favour of the second hypothesis.

A more in-depth investigation is under way into the medicinal practices of local healers to shed light on this issue.

Another way to approach interspecific convergences of plants uses

Rather than considering the question of use convergence from the alternative perspective of knowledge transfers versus convergence of independently acquired uses, a more fruitful approach may be the one that [START_REF] Lestel | Etho-ethnology and ethno-ethology[END_REF] propose under the term ethoethnology. According to the authors this approach involves the study of "the hybrid communities comprised of humans and animals sharing meaning, interests and affect" (Ibid) in which both animals and humans play an active role in the production of knowledge about their environment. [START_REF] Krief | L'interspécificité du pharmakôn dans le parc Kibale (Ouganda) : savoirs partagés entre humains et chimpanzés ?[END_REF] in their study in Kibale National Park in Uganda gives an illustration of such an attempt regarding the particular relationship linking the Batoroo ethnic group and the chimpanzees who share the medicinal use of plant species. The Batoroo's awareness of this knowledge sharing between humans and animals is reflected in the use of a specific term to describe this perceived capacity for "reciprocal imitation between humans and animals" (ibid, our translation). This approach in which mahouts and village elephants would be viewed as a hybrid community seems more appropriate in the context of Thongmyxay district to account for the intertwining that characterizes elephant behaviours, the EURs indicated by mahouts, mahouts own medicinal uses, and the traditional veterinary care they provide to elephants. We have seen for instance in Section 4.5 that some mahouts give their elephants who suffer from diarrhoea the roots that they know it seeks when it experiences this condition.

Convergences identified in other studies

Figure 3F shows the number of mahouts who quoted each species mentioned in the EUR. Table 4 shows ethno-medicinal uses identified and pharmacological properties studied in the literature for species cited in the EUR by at least three mahouts.

The anti-diarrheic property of H. perforata reported [START_REF] Perry | Medicinal plants of East and Southeast Asia: attributed properties and uses[END_REF] is in accordance with EUR and mahouts own uses. However, one mahout uses this item in case of elephant constipation (table 2). An observation consistent with this last use is found in the "elephant care manual for mahouts and camp manager" published by the FAO [START_REF] Phuangkum | Elephant care manual for mahouts and camp managers[END_REF]. In this manual it is reported that elephants purge themselves once or twice a year to remove intestinal parasites using for this purpose different barks and roots, including that of H. perforata. Thus this vegetal item, in addition to its anti-infective properties (table 2), could have a regulatory effect on intestinal transit, as it is the case for instance with Malva sylvestris L. which is reported to be used by humans for both diarrhoea and constipation cases (Jabtri et al., 2017;[START_REF] Barros | Leaves, flowers, immature fruits and leafy flowered stems of Malva sylvestris: A comparative study of the nutraceutical potential and composition[END_REF][START_REF] Dellagreca | Antioxidant and Radical Scavenging Properties of Malva Sylvestris[END_REF]. Surprisingly, we did not find any mention of medicinal uses or properties in the literature for Lithocarpus auriculatus, cited in EUR by seven mahouts, four times in the context of motherhood and three times for diarrhoea.

Conclusion

We have recorded numerous convergences between the observations interpreted by the mahouts as self-medication behaviour on the part of elephants and their own medicinal practices. The validity of these interpretations would require a specific ethological study to be established, but what seems remarkable is the dual interface that is at work around a forest environment common to elephants and humans. Village elephants standing at the first interface between the plants they select and the mahouts who observe them become a source of knowledge for humans. The mahouts stand at the second interface between elephants and the human culture they belong to. It is the place where behavioural elements observed on elephants are integrated into the care provided to them and into mahouts' own medicinal practices.

Our initial concern was to better understand how the observation of animal self-medication behaviour has contributed to human medicinal practices. Mahouts' practices in Thongmyxay district show that beyond a mere reproduction of these animal behaviour observations, the human or veterinary medicinal practices from which they may have been derived are the result of complex arrangements, in which all available medicinal and conceptual resource are integrated.

The current trend in Laos to transfer village elephants to elephant resorts leads to a disruption in the transmission of mahouts' family knowledge [START_REF] Suter | Population viability of captive Asian elephants in the Lao PDR[END_REF]. It is therefore urgent to continue to collect this knowledge and in particular traditional veterinary care in order to transmit it to the new generation of mahouts employed in these resorts and to the people responsible for the health of captive elephants.

Fig. 1 :BFig. 2 :AFig. 3 :

 123 Fig. 1: Contextual maps of Thongmyxay district and location of the ECC centre (Juan A. Torres) wide view (A) close-up view (B)

Acknowledgments

The authors thank the staff working at ECC for their valuable cooperation, and all people interviewed in Thongmyxay district. We are grateful to Elizabeth Elliott and David Wharton for illuminating discussion and editing, and to Juan Antonio Torres for maps. This work was supported by Ekhagastiftelsen Foundation and the Fondation Ensemble.

Diarrhoea

Root of Lasia spinosa (L.) Thwaites with Tinosposra crispa stem in equal quantity are sliced, dried and then pulverized in a mortar, a little honey is added for palatability. The mixture is dried out and given in a rice ball one time a day for 2-3 days.

Unhusked rice is given with salt and elephant is brought daily to a swampy place where it can eat mud.

Steamed leaves of Bischofia javanica

Blume with salt added are given to eat.

Chopped barks of H. perforata and Terminalia mucronata mixed with steamed bitter leaves and salt are given to eat.

Other digestive disorders

Constipation: Root of Lasia spinosa is sliced, dried and then crushed with unhusked rice in equal parts. A ball of the size of a fist is given 2 times a day.

Constipation: Root of Harrisonia perforata is boiled and then mashed in the decoction, salt is added and the mixture is given to the elephant.

Bloating: Root of Lasia spinosa is mixed in equal parts with the fruit of toona sp., husked rice powder and rice bran. Water is added to form a paste. A ball the size of a fist is given 2 times a day until healing.

Abscess

Abscess is lotionned with a decoction of barks of Pterocarpus macrocarpus Kurz and of another tree not identified (mai kao).

Bark of Dillenia parviflora Griff. with salt is given to eat.

Grilled unhusked rice is applied on abscess.

Skin outbreak

A piece of the juicy stem of Entada rheedii Spren. is crushed with a mallet and twisted in water with detergent added. Spots are lotioned with the stem dipped into the preparation.

Chopped Alpinia galanga (L.) Willd. stems are mixed with elephant urine and applied on the affected area 2-3 times a day until healed.

Crushed fruit of

Spondias pinnata combined with human urine is then applied to the skin.

Wounds

A decoction of barks of Cratoxylum formosum Jack Dyer, Pterocarpus macrocarpus, Bauhinia malabarica Roxb. (10kg each) and fruits of Spondias pinnata (4-5 kg), is prepared with 10 liters of water, until it reduces to 2 liters. A stem of Entada rheedii, crushed with a mallet is twisted in preparation and used to lotion the wounds twice a day until cured.

Urine and dry elephant faeces are mixed with crushed Alpinia galanga (in equal amounts) and applied by hand on the wound.

A decoction of sliced stem of Acacia caesia (500g for 2l of water) is used to lotion the wound everyday.

A mixture of clay and elephant faeces is placed in a squame of banana stem and applied as a poultice on wounds.

Apathetic state

Root and stem of Tinospora crispa, dried or fresh is crushed with salt and given to eat.

Table 3: Ethnoveterinary preparations given to elephants.

Plant species

Traditional uses Pharmacological activity

Harrisonia perforata

Root is used as a remedy for treatment of wound healing, diarrhoea, dysentery and cholera [START_REF] Perry | Medicinal plants of East and Southeast Asia: attributed properties and uses[END_REF], and as antitoxic [START_REF] Libman | Medicinal plants: An important asset to health care in a region of Central Laos[END_REF] and fever [START_REF] Maneenoon | Ethnomedicinal plants used by traditional healers in Phatthalung Province, Peninsular Thailand[END_REF] Anti-pyretic, anti-diarrhoea properties [START_REF] Laopaksa | Preliminary study on antibacterial action of Thai medicinal plants for respiratory tract infection (I)[END_REF]cited in Batm et al., 2011). Root inhibits Staphylococcus aureus, Streptococcus pyrogenes [START_REF] Batm | Antimicrobial Activity of the Extracts from Benchalokawichian Remedy and Its Components[END_REF], Mycobacterium smegmatis [START_REF] Chea | In vitro Antimicrobial Activity of Plants used in Cambodian Traditional Medicine[END_REF]. Leaves have anti Plasmodium falciparum activity [START_REF] Nguyen-Pouplin | Antimalarial and cytotoxic activities of ethnopharmacologically selected medicinal plants from South Vietnam[END_REF]. Wood showed activity against HIV-1 IN [START_REF] Bunluepuech | Anti -HIV-1 integrase activity of Thai Medicinal Plants 4[END_REF].

Heteropanax fragrans

Bark is used in a preparation for diarrhoea root and bark are included in another preparation given in case of jaundice [START_REF] Borah | Traditional medicine in the treatment of gastrointestinal diseases in Upper Assam[END_REF] Tinospora crispa Stem is used to promotes blood flow, as appetizer, to reduce fever, body heat and thirst, against diabetes, [START_REF] Maneenoon | Ethnomedicinal plants used by traditional healers in Phatthalung Province, Peninsular Thailand[END_REF] Antimalarial activity [START_REF] Nik A Rahman | Antimalarial activity of extracts of Malaysian medicinal plants[END_REF], antinociceptive and anti-inflammatory activities [START_REF] Sulaiman | Antinociceptive and anti-inflammatory activities of Tinospora crispa in various Animal models[END_REF], decreased blood glucose and increased insulin levels in diabetic rats (Noor and Ashscoff, 1989)

Bark is used in case of sore teeth [START_REF] Khuankaew | Ethnobotanical study of medicinal plants used by Tai Yai in Northern Thailand[END_REF]. Wood is used in a preparation for leucorrhea and infertility due to menstrual disorders [START_REF] Chotchoungchatchai | Medicinal plants used with Thai Traditional Medicine in modern healthcare services: A case study in Kabchoeng Hospital, Surin Province, Thailand[END_REF] Stem extract has shown cytotoxicity against HepG2 cells [START_REF] Weerapreeyakul | Evaluation of the anticancer potential of six herbs against a hepatoma cell line[END_REF]

Castanopsis indica

Leaves are used to treat stomach disorder and skin diseases. Plant resin is given to treat diarrhoea. A paste of leaves is applied for headache [START_REF] Malla | Indigenous knowledge on ethnobotanical plants of Kavrepalanchowk district[END_REF]. Bark paste is applied on chest to control chest pain [START_REF] Joshi | Indigenous knowledge and uses of medicinal plants in Macchegaun, Nepal[END_REF] Free radical scavenging activity of leave extract could have potential hepatoprotective properties [START_REF] Dolai | Free radical scavenging activity of Castanopsis indica in mediating hepatoprotective activity of carbon tetrachloride intoxicated rats[END_REF])

Maclura cochinchinensis

Root is used to cure malaria and colds [START_REF] Kobayashi | Indonesian Medicinal Plants. XXI. Inhibitors of Na+/H+ Exchanger from the Bark of Erythrina variegata and the Roots of Maclura cochinchinensis[END_REF]. Heartwood used to treat chronic fever, skin infection, abnormality of the lymph node, diarrhoea, fainting, skin infection; [START_REF] Leakaya | Antioxidant Activity, Total Phenolic, Total Flavonoid Content and HPTLC Analysis of Morin in Maclura cochinchinensis Heartwood Extract[END_REF] Extract from heartwood showed antibacterial activity against Staphilococcus aureus, Staphylococcus epidermidis, Bacillus subtilis ; anti-fungal activity (dermatophytes) and antiherpes simplex virus (HSV) activity [START_REF] Kongkiatpaiboon | Determination of Morin in Maclura cochinchinensis Heartwood by HPLC[END_REF].

Phyllanthus reticulatus

Leaves and bark are used as astringent and diuretic. Juice of leaves is used for the treatment of diarrhoea in children [START_REF] Ghani | Medicinal Plants of Bangladesh with Chemical Constituents and Uses[END_REF] The bark showed antiviral [START_REF] Saha | The Analgesic and Anti-Inflammatory Activities of the Extracts of Phyllanthus reticulatus. in Mice Model[END_REF] and antiplasmodial activity [START_REF] Omulokoli | Antiplasmodial activity of four Kenyan medicinal plants[END_REF]. Aerial parts Aerial parts extract shows analgesic and anti-inflammatory properties [START_REF] Saha | The Analgesic and Anti-Inflammatory Activities of the Extracts of Phyllanthus reticulatus. in Mice Model[END_REF].

Cratoxylum formosum

The bark, root and leaves are used in folk medicines to treat fevers, coughs, diarrhoea, itches, ulcers and abdominal complaints [START_REF] Nguyen | Triterpenoid and xanthone constituents of Cratoxylum cochinchinense[END_REF] Xanthones extracted from root has shown antibacterial and antifungal activity and cytotoxic activity [START_REF] Boonsri | Antibacterial and cytotoxic xanthones from the roots of Cratoxylum formosum[END_REF][START_REF] Boonnak | Chromene and Prenylated Xanthones from the Roots of Cratoxylum formosum ssp[END_REF] Table 4: Some ethno-medicinal uses and properties found in the literature for the species cited by at least three mahouts. Convergences with EUR on plant parts or indications are highlighted in bold font.

Mahout interview

Mahout medicinal practice Elephant plant selection Specimen collection with mahout

LAOS

Elephant/mahout plant use convergence


Ophélie Gervais

Bruno Chollet

Christine Dubreuil

Serena Durante

Chunyan Feng

Cyril Hénard

Cyrielle Lecadet

Delphine Serpin

Renault Tristan

Isabelle Arzul
email: isabelle.arzul@ifremer.fr

Gervais Ophélie

Chollet Bruno

Durante Serena

Feng Chunyan

Hénard Cyril

Lecadet Cyrielle

Serpin Delphine

Tristan Renault

Arzul Isabelle

Involvement of apoptosis in the dialogue between the parasite Bonamia ostreae and the flat oyster Ostrea edulis

Keywords: Apoptosis, Ostrea edulis, Bonamia ostreae, flow cytometry, gene expression, mollusk

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

Introduction

Populations of the European flat oysters, Ostrea edulis, have suffered from overfishing and several diseases including bonamiosis due to the parasite Bonamia ostreae. This protozoan was first detected in association with mortality of flat oysters in 1979 in Brittany [START_REF] Pichot | Recherches sur Bonamia ostreae gen. n., sp. n., parasite nouveau de l'huitre plate Ostrea edulis L[END_REF] and was subsequently identified in other production sites in Europe. It is currently reported in several other European countries, in North America and New Zealand where it has recently been detected in another oyster species Ostrea chilensis [START_REF] Lane | Bonamia ostreae in the New Zealand oyster Ostrea chilensis: a new host and geographic record for this haplosporidian parasite[END_REF].

Mortality due to B. ostreae mostly affects oysters older than two years although the parasite infects all developmental stages including larvae and spat [START_REF] Arzul | Can the protozoan parasite Bonamia ostreae infect larvae of flat oysters Ostrea edulis?[END_REF][START_REF] Balouet | Haemocytic parasitosis: Morphology and pathology of lesions in the French flat oyster, Ostrea edulis[END_REF][START_REF] Grizel | Etude des recentes epizooties de l'huitre plate Ostrea Edulis Linne et de leur impact sur l'ostreiculture bretonne[END_REF][START_REF] Lynch | The susceptibility of young prespawning oysters, ostrea edulis, to bonamia ostreae[END_REF]. Transmission of the parasite is direct from infected to non-infected oysters and does not seem to require intermediate host [START_REF] Culloty | Susceptibility of a number of bivalve species to the protozoan parasite Bonamia ostreae and their ability to act as vectors for this parasite[END_REF]. The parasite infects and multiplies within hemocytes and can be observed extracellularly in epithelium [START_REF] Comps | Etude ultrastructurale d'un protiste parasite de l'huître Ostrea edulis[END_REF][START_REF] Pichot | Recherches sur Bonamia ostreae gen. n., sp. n., parasite nouveau de l'huitre plate Ostrea edulis L[END_REF]. Routes of entrance and release of the parasite in the oysters remain undetermined. However, several studies have suggested the role of the gills in the infection and excretion of B. ostreae [START_REF] Comps | Recherches histologiques et cytologiques sur les infections intracellulaires des mollusques bivalves marins[END_REF]. Although the parasite is not cultivable, the establishment of a protocol to purify B. ostreae from infected oysters [START_REF] Mialhe | Isolation and purification of the protozoan Bonamia ostreae (Pichon et al. 1980), a parasite affecting the flat oyster Ostrea edulis[END_REF] has allowed the development of in vitro and in vivo experimental infection protocols [START_REF] Hervio | Establishment of an experimental infection protocol for the flat oyster, Ostrea edulis, with the intrahaemocytic protozoan parasite, Bonamia ostreae: application in the selection of parasite-resistant oysters[END_REF][START_REF] Mourton | Interactions between Bonamia ostreae (Protozoa: Ascetospora) and hemocytes of Ostrea edulis and Crassostrea gigas (Mollusca: Bivalvia): in vitro system establishment[END_REF]. In vitro infection experiments consisting in contact between hemocytes and parasites allow investigating host parasite interactions at the cellular level and have been used to better understand mechanisms involved in the resistance of the flat oyster to B. ostreae infection. Previous studies have demonstrated that both hemocytes and B. ostreae are actively involved in the internalization of the parasite [START_REF] Chagot | Interactions between Bonamia ostreae (Protozoa: Ascetospora) and hemocytes of Ostrea edulis and Crassostrea gigas (Mollusca: Bivalvia): Entry mechanisms[END_REF][START_REF] Prado-Alvarez | Heat Shock Protein 90 of Bonamia ostreae: Characterization and Possible Correlation with Infection of the Flat Oyster, Ostrea edulis[END_REF] and have notably showed the involvement of apoptosis in the defence against bonamiosis [START_REF] Gervais | Molecular and cellular characterization of apoptosis in flat oyster a key mechanisms at the heart of host-parasite interactions[END_REF][START_REF] Gervais | Flat oyster follows the apoptosis pathway to defend against the protozoan parasite Bonamia ostreae[END_REF].

Apoptosis is a universal mechanism involved in different biological processes including defense against stressors like pathogens [START_REF] Hervio | Establishment of an experimental infection protocol for the flat oyster, Ostrea edulis, with the intrahaemocytic protozoan parasite, Bonamia ostreae: application in the selection of parasite-resistant oysters[END_REF][START_REF] Sokolova | Apoptosis in molluscan immune defense[END_REF]. By degrading infected cells, apoptosis contributes indeed to limit pathogen multiplication and spread [START_REF] Sokolova | Apoptosis in molluscan immune defense[END_REF]. However, parasites have developed in turn strategies to modulate host cell apoptosis to successfully install into their hosts. Modulation of apoptosis appears as an important weapon in the pathogenic arsenal of multiple intracellular protozoan parasites including Leishmania major, L. Mexicana and L. amazonensis which inhibits macrophage apoptosis by inducing PI3-kinase/Akt a signalling pathway known to promote cell survival [START_REF] Ruhland | Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis[END_REF].

In vitro experiments have also previously suggested that B. ostreae inhibits hemocyte apoptosis after 4 h of contact notably by increasing overexpression of an inhibitor of apoptosis (IAP) [START_REF] Morga | New insights in flat oyster Ostrea edulis resistance against the parasite Bonamia ostreae[END_REF]. By simplifying the conditions of infection, in vitro experiments allow identifying most distinct features of the response of the oyster. However, these responses might be mitigated by other processes at the oyster scale and relative importance of these features needs to be appreciated at the animal level through in vivo trials.

Infection experiment by injecting parasites into the adductor muscle of the oyster allows subjecting oysters to a similar dose of pathogens and following the response of the oysters in time. Such approach has previously showed that infection with B. ostreae is associated with a decrease of phagocytosis and ROS production in «resistant» oysters contrary to wild oysters [START_REF] Morga | Contribution of in Vivo Experimental Challenges to Understanding Flat Oyster Ostrea edulis Resistance to Bonamia ostreae[END_REF]. Furthermore, the response against bonamiosis is known to differ according to oyster populations [START_REF] Harrang | Apport des informations moléculaires et cellulaires pour la caractérisation de la résistance de l'huître plate européenne vis-à-vis de la bonamiose, et pour la détection de signatures de la sélection naturelle[END_REF].

In this context, the relative involvement of apoptosis in the dialogue between flat oyster and B. ostreae has been investigated in vivo on two genetically distinct oyster populations, from Quiberon bay in Brittany and from Corsica in South of France [START_REF] Harrang | Apport des informations moléculaires et cellulaires pour la caractérisation de la résistance de l'huître plate européenne vis-à-vis de la bonamiose, et pour la détection de signatures de la sélection naturelle[END_REF]. Apoptosis was evaluated during an experimental infection using flow cytometry and molecular tools previously developed [START_REF] Gervais | Induction of apoptosis by UV in the flat oyster, Ostrea edulis[END_REF].

Material and method

Flat oysters

Two year flat oysters, O. edulis, were collected from Quiberon bay (Brittany, France) and Etang de Diana (Corsica, France) and acclimatized over 30 days in Ifremer's Facilities.

Oysters were maintained in 150 L raceway supplied with a constant flow of seawater enriched in phytoplankton (Skeletonema costatum, Isochrysis galbana and Tetraselmis suecica).

Parasite isolation

Parasites were purified according to the protocol developed by [START_REF] Mialhe | Isolation and purification of the protozoan Bonamia ostreae (Pichon et al. 1980), a parasite affecting the flat oyster Ostrea edulis[END_REF].

Examination of gill tissue imprints in light microscopy allowed selecting oysters highly infected with B. ostreae. All organs except the adductor muscle were homogenized and purified by differential centrifugation on sucrose gradients. Bonamia ostreae cells were washed and resuspended in filtered sea water (FSW) and counted with hemocytometer.

Experimental infection

The experimental design consisted of 10 tanks (5 for oysters injected with parasites and 5 for oysters injected with FSW). Each tank contained 30 oysters from Quiberon Bay and 30 oysters from Corsica (Figure 1). Purified parasites (7.5.10 5 per individual) were injected into 150 oysters from Quiberon and 150 oysters from Corsica. The same amount of oysters was injected with 100 µl of FSW. Injection was performed into the adductor muscle after anesthesia using MgCl 2 (250g) in 4 L of tap water and 1 L of seawater supplemented in phytoplankton. Flat oysters were maintained for one month in 150 L tank with constant flow of seawater enriched in phytoplankton. Potential mortality was checked daily.

Hemolymph collection

Ten oysters were collected from each tank at 1, 4, 7 30 and 34 days after injection.

Hemolymph was withdrawn from the adductor muscle of the oysters using 1 mL syringe.

Hemolymphs from 10 oysters subject to similar experimental conditions were pooled and kept on ice to avoid cellular aggregation. After filtration at 70 µm to remove debris, hemocyte concentration was estimated using a hemocytometer and adjusted at 5.10 5 cell.ml -1 with FSW for flow cytometry, estimation of DNA fragmentation and real time PCR.

Flow cytometry

Modulation of intracytoplasmic calcium concentration was monitored using Fluo-4/AM (Molecular Probes). Modulation of mitochondrial membrane potential (ΔΨm) was measured using JC-10 dye (FluoProbes®) and externalization of phosphatidyl serine on plasma membrane and cell viability were tested using Annexin V and PI respectively (Eurobio).

These activities were monitored by flow cytometry using an EPICS XL 4 (Beckman coulter) according to [START_REF] Gervais | Induction of apoptosis by UV in the flat oyster, Ostrea edulis[END_REF]. Six replicates were tested for each condition and at each sampling time.

Real time PCR for Bonamia ostreae detection

A piece of gill was sampled from 5 individuals per tank and time of sampling and fixed in 90% ethanol. Gill tissue (25 mg) was collected for DNA extraction using the QiaAmp DNA Mini-kit (Qiagen) according to the manufacturer's instructions. DNA was eluted and resuspended in 50 µl of sterile, deionized water and then diluted to a final concentration of 5 ng.µl -1 for real time PCR analysis.

Real time PCR was performed using a MX3000 Thermocycler sequence detector (Stratagene) according to a previously published protocol Robert et al. (2009). All reactions were carried out in duplicate. Each PCR run included negative controls consisting in reaction mixtures without DNA and positive control consisting of plasmidic DNA including the target region.

Gene expression analysis

RNA isolation and cDNA synthesis

Samples of hemolymph were centrifuged at 4°C during 10 min at 1500 x g and the supernatant was discarded and kept at -80°C in TRIzol® Reagent (Ambion) until being processed. Total RNA was extracted using TRIzol® Reagent according to manufacturer's recommendations. Total RNA was treated with TurboTM DNase (Ambion) to remove genomic DNA. RNA concentration and quality was estimated using the NanoDrop 2000 (Thermo Scientific) and Bioanalyser 2100 (Agilent). Direct real time PCR with no retrotranscription was performed using EF1-α primers (Table 1) after each DNase treatment to control absence of genomic DNA. First strand cDNA synthesis was carried out from 500 ng of RNA treated using the SuperScript®III First-Strand Synthesis System (Invitrogen).

Real time quantitative PCR

Expression level of O. edulis apoptotic-related genes previously described in [START_REF] Gervais | Molecular and cellular characterization of apoptosis in flat oyster a key mechanisms at the heart of host-parasite interactions[END_REF] was measured in each pool for both oyster groups after B. ostreae or FSW injection at each time of the experiment. Real time quantitative PCR was carried out in duplicate in 96-microwell plates using Mx3000 Thermocycler sequence detector (Stratagene). Amplification reactions contained 2 µL of each primer, 10 µL of Brillant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent Technologies) and 5 µL of diluted cDNA (1/30) in a final volume of 20 µL. Amplification was carried our using standard cycling conditions: 95°C for 3 min, followed by 40 cycles of 95°C for 5 s and 60°C for 10 s. Efficacies were previously calculated for each primer pair in Article 3 (Table 9). Expression level of selected genes was analysed using the method described by Pfaffl, (2001). Host gene expression was normalized using elongation factor-1α (EF-1α) according to [START_REF] Morga | Identification of genes from flat oyster Ostrea edulis as suitable housekeeping genes for quantitative real time PCR[END_REF]. Calibrators consisted of samples injected with FSW at each time of the experiment.

In addition, expression level of B. ostreae 18s was followed in oysters from Quiberon after parasite injection and in control condition. Real time quantitative PCR was carried out as previously described by Pfaffl, (2001).

Statistical analyses

Normality of all data sets was tested using Shapiro Wilk test and homogeneity of variances were assumed using Bartlett's and Levene test. Two-way ANOVA were used for normal data sets to determine whether significant differences existed between time and experimental treatments. Tukey's post hoc test was used to establish where significant differences occurred within the data set. Non normal distribution data sets were analysed using Wilcoxon test.

Analyses were conducted using RStudio.

Results

Parasite detection

Parasite detection was investigated at the individual level in gills and in pool of hemocytes using real time PCR.

Parasite DNA in gills

No mortality was observed in challenged oysters during the experiment.

Quiberon bay is an endemic zone regarding infection with B. ostreae. The parasite was detected in up to 30% of tested oysters injected with FSW. B. ostreae was detected in 80% and then less than 50% of tested Quiberon oysters injected with parasites at 7 days and after day 30 respectively (Figure 2A).

In contrast to Quiberon, prevalence of bonamiosis in Corsica is known to be very low and Corsican oysters injected with FSW appeared negative by PCR (Figure 2B). Up to 60% of tested Corsican oysters injected with parasites were detected positive 1 and 30 days after injection.

Parasite RNA in hemolymph

To facilitate the reading of Figure 3, results were represented by 40 (maximum ct value) -Ct value of sample.

B. ostreae 18s RNA was followed in order to quantify parasite infection in hemolymph collected from Quiberon oysters injected with FSW (Figure 3B) and from all the oysters injected with parasite (Figures 3A-C). Whatever the tested condition was, B. ostreae RNA was detected as soon as one day after injection. In Quiberon oysters, same amount of parasite RNA was detected when injected with parasites and with FSW. Less parasite RNA was detected in Corsican oysters injected with the parasite than in Quiberon oysters whether they were injected with parasites or FSW (p<0.05) (Figure 3C). Dynamics of B. ostreae 18s RNA was similar in oysters injected with parasites whatever their origin was, decreasing from day 1 to day 30.

Modulation of hemocyte cellular activities

Involvement of apoptosis response of flat oysters during an infection with B. ostreae was investigated at the cellular level by measuring by flow cytometry the intracytoplasmic calcium concentration, the mitochondrial membrane potential and the externalization of phosphatidyl serine on plasma membrane.

Whatever the origin was, injection of B. ostreae induced a significant decrease of cells with high intracytoplasmic calcium concentration compared to the control 7 and 34 days after injection (Figure 4A). Modulation of this parameter was not significant at days 1, 4 and 30.

In oysters from Quiberon, percentages of hemocytes with low ΔΨm appeared slightly modulated compared to the control (Figure 4B). In contrast, this parameter was significantly increased in Corsican oysters 1, 7 and 34 days after injection and decrease at day 30.

In oysters from Quiberon percentage of cells with externalization of phosphatidyl serine in oyster from Quiberon was higher only after 4 days of infection (Figure 4C). In contrast Corsican oysters showed a decrease of cells with externalization of phosphatidyl serine compared to the control at 4 and 7 days post infection.

Ostrea edulis gene expression

Involvement of apoptosis in the response of flat oysters during an infection with B. ostreae was investigated at the molecular level by measuring the expression level of seven genes involved in both the extrinsic and intrinsic apoptotic pathways. These genes were chosen to provide an overview of the apoptotic response of the flat oyster.

Extrinsic pathway

Five genes involved in the extrinsic pathway were followed during the experiment: TNFL, TNFR, TTRAP, caspase 2 and caspase 3.

In oysters from Quiberon injected with parasites, first significant gene expression modulation was observed at day 4 with an activation of caspase 3 which was followed at day 7 by an overexpression of TNFL and TTRAP (Figures 5A,C,H). 30 days post injection, TNFR and caspase 3 were down regulated. Finally at day 34, caspase 2 appeared overexpressed.

In Corsican oysters injected with B. ostreae, as soon as 1 day post injection TNFL and TNFR appeared under expressed and caspase 3 over expressed compared to the control (Figures 5A,B,E,H). TNFL expression increased at day 4 and was under expressed 30 days post injection like TTRAP and caspase 2. Finally, caspase 3 appeared overexpressed at day 34 compared to the control.

Intrinsic pathway

Two genes involved in the intrinsic pathway were followed during the experiment: Bcl2 and endoG. Bcl2 is involved in the inhibition of apoptosis by the mitochondria and endoG is an enzyme that can induce DNA fragmentation.

In oysters from Quiberon, these two genes were upregulated 7 post injection with parasites and endoG appeared then under regulated at 30 days (Figure 5F-G,H). In contrast, in Corsican oysters these two genes were under expressed after 30 days and endoG was overexpressed after 34 days.

Discussion

Successful establishment of infections in susceptible hosts requires means of avoiding host responses soon after initiating infection [START_REF] Yoshino | Parasite-Invertebrate Host Immune Interactions[END_REF]. Installation of the parasite Bonamia ostreae into its host, the flat oyster Ostrea edulis seems to be a slow process.

Experimental infections by cohabitation with naturally infected oysters showed that parasite detection was observed after 3 months in non-infected ones and first mortality was reported after 7 months [START_REF] Elston | Occurence and significance of bonamiosis in european flat oysters ostrea edulis in north America[END_REF]. In contrast, when the parasite source consisted of oysters previously injected with high doses of B. ostreae, first mortality was observed more quickly, after 2 months [START_REF] Morga | Contribution of in Vivo Experimental Challenges to Understanding Flat Oyster Ostrea edulis Resistance to Bonamia ostreae[END_REF]. Injection of 50% ID (infectious dose) induced first mortality after 4 months [START_REF] Hervio | Establishment of an experimental infection protocol for the flat oyster, Ostrea edulis, with the intrahaemocytic protozoan parasite, Bonamia ostreae: application in the selection of parasite-resistant oysters[END_REF]. Moreover, with higher parasite doses, B. ostreae can be detected as soon as first day after injection [START_REF] Morga | Contribution of in Vivo Experimental Challenges to Understanding Flat Oyster Ostrea edulis Resistance to Bonamia ostreae[END_REF]. Moreover, previous studies have confirmed involvement of apoptosis in the response of the flat oyster, O. edulis, to in vitro infection by the parasite B. ostreae [START_REF] Gervais | Molecular and cellular characterization of apoptosis in flat oyster a key mechanisms at the heart of host-parasite interactions[END_REF][START_REF] Gervais | Flat oyster follows the apoptosis pathway to defend against the protozoan parasite Bonamia ostreae[END_REF].

Considering these results, we have investigated response mechanisms and more particularly the apoptotic response of the oyster in the early and later phases of the infection after injection.

Experimental infection was carried out by injecting a fixed amount of parasites in the sinus of the adductor muscle of the oysters. Although injection might induce some stress and tissue lesions, no mortality was observed during the course of the experiment. Flat oysters from two natural locations were challenged concurrently in order to investigate the specificity of the response against B. ostreae. These populations were selected for their genetic differences demonstrated using microsatellite and SNP markers [START_REF] Harrang | Apport des informations moléculaires et cellulaires pour la caractérisation de la résistance de l'huître plate européenne vis-à-vis de la bonamiose, et pour la détection de signatures de la sélection naturelle[END_REF] and their prevalence regarding bonamiosis. Quiberon bay is an endemic location regarding bonamiosis with prevalence generally between 10 and 15 % [START_REF] Arzul | Bonamia ostreae and Ostrea edulis: a stable host-parasite system in France[END_REF]. In contrast, prevalence in Corsica is low and associated with a congener species, Bonamia exitiosa [START_REF] Arzul | Distribution and variability of bonamia exitiosa in flat oyster ostrea edulis population in Europe[END_REF][START_REF] Arzul | Etude de la résistance de la population d'huîıtres plates Ostrea edulis dans l'étang de Diana au parasite Bonamia ostreae[END_REF].

Parasite DNA was detected in gills of oysters from both populations after parasite injection but also in control oysters from Quiberon. These results are concordant with prevalence of bonamiosis in both populations. Oysters from Quiberon showed a peak of detection 7 days after injection whereas Corsican individuals presented a peak of parasite DNA detection 1 day and then 30 days after injection. However, PCR allows detecting parasite DNA whether the parasite is alive or dead. In contrast, presence of RNA is indicative of live stages and live B. ostreae could be detected all sampling days in some pools of oysters injected with the parasite. Early detection of parasite DNA and RNA might correspond to injected parasites and, in Quiberon oysters, also to previously established infection. Later detection at days 30 and 34 showed that B. ostreae successfully installed in oysters from both populations.

Previous studies also reported that the installation of the parasite into its host was effective around 30 days after injection [START_REF] Morga | Contribution of in Vivo Experimental Challenges to Understanding Flat Oyster Ostrea edulis Resistance to Bonamia ostreae[END_REF].

Apoptosis response to B. ostreae was followed at the cellular and molecular levels using tools previously developed. Intracytoplasmic calcium concentration was reduced in both populations at 7 and 30 days post infection. It was previously showed that in vitro infection with the parasite also induced a decrease of intracytoplasmic calcium [START_REF] Gervais | Flat oyster follows the apoptosis pathway to defend against the protozoan parasite Bonamia ostreae[END_REF].

Calcium is important in cytoskeleton reorganization during parasite internalization [START_REF] Epting | Molecular mechanisms of host cell invasion by Trypanosoma cruzi[END_REF]. This reduction could be due to an increase of internalization of B. ostreae by the hemocytes. The other tested cellular parameters showed differences between oyster populations. Oysters from Quiberon, showed indeed an increase of annexin V labelled cells at day 4 whereas this parameter was downregulated in Corsican oysters compared to the control at day 7. Finally, mitochondrial membrane potential appeared more modulated in Corsican than in Britain oysters. Presence of parasites into hemocytes was associated with an increase of some cellular tested activities but B. ostreae installation appeared to inhibit apoptosis induction. It was previously showed that B. ostreae can inhibit apoptosis in flat oyster hemocytes in vitro after 44 h [START_REF] Gervais | Molecular and cellular characterization of apoptosis in flat oyster a key mechanisms at the heart of host-parasite interactions[END_REF].

Expression level of apoptotic genes as modulated differently in both populations after parasite injection. For oysters from Quiberon, the apoptotic response seemed to be activated at 7 days post-injection whereas after 30 days it appeared inhibited. In Corsican oysters, the apoptotic response was inhibited 30 days after injection, and, in contrast, activated after 34 days. For oysters from Quiberon, apoptosis activation observed at day 7 can be related to the high detection of parasites in gills. In contrast, inhibition of apoptosis in both groups may be associated with parasite installation in the flat oysters as observed at day 30 or decrease of parasite RNA detected on oyster hemolymph. Apoptotic parameters monitored in the context of this study were modulated by the injection of parasites. However, they did not fluctuate all in the same way in the same time and, interestingly, were modulated differently in oysters originating from both oyster groups. Indeed, infection with B. ostreae develops differently between each individual and does not occur in the same way for both groups and can influence the apoptotic response. Our results confirm that interactions between parasite and their host are complex. Moreover, apoptosis is a process that can take hours or days to conduct to the cell death from the activation of initial trigger but once the mitochondria is compromised the cell death is very quick [START_REF] Green | Apoptotic Pathways: Ten Minutes to Dead[END_REF]. This pathway is the result of multiple activation and inhibition mechanisms that do not occur in the same time. Our experiments suggest that apoptosis is deeply involved in the response of the oyster against B. ostreae and differently depending on the stage of the infection. This balance between activation and inhibition of apoptosis might reflect the battle between the oyster and its parasite.

Figure 1 .

 1 Figure 1. Experimental design of infection by injection.

Figure 2 .

 2 Figure 2. Number of oysters detected positive by PCR in gills after injection with filtered sea water (FSW) or parasite (Bo) from Quiberon (A) and Corsica (B) at different times. n=15.

Figure 3 .

 3 Figure 3. Detection of Bonamia ostreae 18s rRNA in pools of hemolymph from Quiberon oysters injected with B. ostreae (A), injected with FSW (B) and Corsican oysters injected with B. ostreae (C) at different times. n= 6 (3 pools per time, 2 replicates per pool).

Figure 4 .

 4 Figure 4. Intracytoplasmic calcium concentration in hemocytes of flat oyster from Quiberon and Corsica (A). Ratios of labeled hemocytes with high calcium concentration between oysters injected with B. ostreae and with FSW. Mitochondrial membrane potential (ΔΨm) in hemocytes of flat oyster from Quiberon and Corsica (B). Ratios of labeled hemocytes with low ΔΨm between oysters injected with B. ostreae and with FSW. Plasma membrane integrity in hemocytes of flat oysters from Quiberon and Corsica (C). Ratio of AnnexinV positive and PI negative hemocytes between oysters injected with B. ostreae and with FSW.Results represent the mean ± SD. n=6.

Figure 5 .

 5 Figure 5. Gene expression in Quiberon and Corsican oysters after parasite injection at different times. Relative expression by quantitative PCR of genes involved in the extrinsic pathway, TNFL (A), TNFR (B), TTRAP (C), Caspase 2 (D), Caspase 3 (E) and in the intrinsic pathway, Bcl2 (F), endoG (G). Expression levels were normalized to EF1-α and presented as relative expression to controls injected with FSW. * p<0.05. Results represent the mean ± SD. n=3. Heatmap of relative expression of apoptotic genes in Quiberon and Corsican oysters (H). The colour legend represents genes expression from violet (under expression) to yellow (over expression).

Table 1 .

 1 Primer sequences and characteristics for quantitative real time PCR.

Table 1 .

 1 Primer sequences and characteristics for quantitative real time PCR.

	Species	Gene	Sequences 5'-3'	Primer	Amplicon	Tm (°C) Efficacies	Reference
				concentration	length (bp)		(%)
				(µM)
	O. edulis	Bcl2	Forward TCCAGACTCGGAAGAAAGGA	4	263	84,3	96,3	Cahais et al., 2012
			Reverse GGGGAAGGATGAATGGAGAT	4
		casp 2	Forward ATGACGGTGAGGAACAGGTC	3	242	81,2	98,2	Cahais et al., 2012
			Reverse GGATTCCGAATGGCCTTAAT	3
		casp3	Forward GCAGTTTTTCCGGAATGAGA	3	188	81,7	97,3	Cahais et al., 2012
			Reverse CCGATTCACTTGAGTGAGCA	3
		TNFL	Forward TTCCTAACGTCCCGAACAAC	4,5	224	84,3	97,6	Cahais et al., 2012
		11
			Reverse GCCACTCAGTCATCCTCCAT	4,5
		TNFR	Forward ATGGCGTCCCAATTAGTCTG	4	232	84,3	101,9	Cahais et al., 2012
			Reverse TTCGGTTCCAAATCTTCGTC	4
		endoG	Forward TCCATCATCAATCGAACGAA	5	186	81,5	95	Cahais et al., 2012
			Reverse GCCGATACCCTTCAAAAACA	5
		TTRAP Forward AGTTAAAGCTGCTGCACTACACC 4	234	83,8	96,8	Cahais et al., 2012
			Reverse CTGGAATGCCTTAGATACAATCG 4
		EF1-α	Forward GTCGCTCACAGAAGCTGTACC	4	162	82,9	99	Cahais et al., 2012
			Reverse CCAGGGTGGTTCAAGATGAT	4
	B. ostreae 18s	Forward TCAGCACTTTTCGAGAAATCAA	4,5	200	83	94,6	Cochennec et al.,
			Reverse CCACCATGCATAGAATCAAGAA 4,5				2000

Acknowledgments

This work was supported by Ifremer and the Région of Poitou Charentes, France. The authors thank members of the Ifremer hatchery in La Tremblade, France. A. Langlade and Y. Baldi are also acknowledged for supplying flat oysters from Quiberon and Diana, France.