Jérôme Moreau

Nina Buffenoir

Denis Thiery

Fanny Vogelweith
email: fannyvogelweith@gmail.com

Denis Thiéry

Lobesia botrana as a preferred host of Campoplex capitator, the most occurring larval parasitoid in European vineyards

Keywords: grapevine moth, host species preference, larval parasitoid, choice test

Lobesia botrana as a preferred host of Campoplex capitator, the most occurring larval parasitoid in

European vineyards.

Introduction

With more than 900,000 known species, insect herbivores represent nearly a quarter of all terrestrial macroscopic biodiversity, acting as a dominant component of all terrestrial food webs on Earth (Resh and Cardé 2009;[START_REF] Sauvion | Insect-plant interactions in a crop protection perspective[END_REF]. While they are well regulated by both host plant and natural enemies within their ecosystems [START_REF] Kaplan | Indirect plant-parasitoid interactions mediated by changes in herbivore physiology[END_REF], agroecosystems often break this balancethrough agriculture intensification, monoculture development and pesticide useand favor the emergence of pest species [START_REF] Wilby | Natural enemy diversity and pest control: Patterns of pest emergence with agricultural intensification[END_REF]. Even if only 2% of insect herbivores species are considered as pests [START_REF] Dhaliwal | Insect pest problems and crop losses: changing trends[END_REF], they are responsible for an estimated crop loss of up to 50 % [START_REF] Thacker | A brief history of arthropod pest control[END_REF][START_REF] Aggarwalim | InfoCrop: a dynamic simulation model for the assessment of crop yields, losses due to pests, and environmental impact of agroecosystems in tropical environments. I. Model description[END_REF] and are consequently a major human concern, with implications for public health and agriculture. At a crucial time when farmers struggle to increase or at least maintain yields while reducing pesticide use for environmental and health issues, biological control and integrated pest management strategies appear to be the best current alternatives [START_REF] Brewer | Approaches and incentives to implement integrated pest management that addresses regional and environmental issues[END_REF].

Biological control is based on population regulation and specially on interactions between a hosthere, insect herbivoreand its natural enemies (e.g. parasitoids, predators, pathogens) [START_REF] Van Driesche | Control of pests and weeds by natural enemies[END_REF]. It includes, for example, the release of one or more natural enemies into a targeted agroecosystem to control a growing pest population. Parasitoids specially hymenopterous parasitoidsare key biological agents, and have been the most common type of natural enemy used in crop protection [START_REF] Van Driesche | Control of pests and weeds by natural enemies[END_REF][START_REF] Giunti | Parasitoid learning: Current knowledge and implications for biological control[END_REF]. Parasitoids evolve in a complex environment and therefore rely on a variety of stimuli (e.g. visual, vibrational and olfactory cues) to locate a suitable habitat with host, food and/or mates (see Giunti et al. 2015 for reviews). Multiple factors can influence parasitoids' host foraging, including host size and density, but also importantly their learning capacity [START_REF] Lin | The effect of parasitoid host-size preference on host population growth rates: An example of Aphidius colemani and Aphis glycines[END_REF][START_REF] Giunti | Parasitoid learning: Current knowledge and implications for biological control[END_REF][START_REF] Morgan | Density dependence and environmental factors affect population stability of an agricultural pest and its specialist parasitoid[END_REF]. While there is a heritable component for habitat preference, it remains unclear as to exactly when this preference development occurs (i.e. during early life stages, after emergence, or both), despite numerous theories informing optimal parasitoid foraging [START_REF] Barron | The life and death of Hopkins' host-selection principle[END_REF][START_REF] Davis | The effect of natal experience on habitat preferences[END_REF][START_REF] Giunti | Parasitoid learning: Current knowledge and implications for biological control[END_REF]. The notion of Natal Habitat Preference Induction (NHPI) has been proposed by [START_REF] Davis | The effect of natal experience on habitat preferences[END_REF] to better understand such processes, and states that animals prefer to develop in a similar habitat to the one they experience in early life stage. The learning process associated with this notion is mostly related to chemical cues coming from the natal habitat which are memorized during ontogeny [START_REF] Corbet | Insect chemosensory responses: A chemical legacy hypothesis[END_REF][START_REF] Ruther | Rich in phenomena-lacking in terms. A classification of kairomones[END_REF]. For instance, females of the parasitoid Psyttalia concolor (Hymenoptera: Braconidae) exhibit different oviposition preferences depending on their natal hosti.e. females did not always prefer to oviposit in their natal host [START_REF] Giunti | Early adult learning affects host preferences in the tephritid parasitoid Psyttalia concolor (Hymenoptera: Braconidae)[END_REF]. Altogether, this raises one of the most important challenges in biological control: the plastic specificity between the bio-agent and targeted host.

In order to conduct an efficient and sustainable pest population control, the parasitoid should be able to successfully target its pest species [START_REF] Van Driesche | Control of pests and weeds by natural enemies[END_REF], even in an environment susceptible to host several pest species. Agroecosystems, such as vineyards, can host a large number of pests living in sympatry [START_REF] Vogelweith | Assessing the non-targeted effect of copper on the leaf arthropods communities in a vineyard[END_REF], and as such, investigating host preference in parasitoids is crucial for biological control. The grape berry moth Eupoecilia ambiguella (Hubn.) (Lepidoptera: Tortricidae) and the European grapevine moth Lobesia botrana (Den. & Schiff.) (Lepidoptera: Tortricidae) are two polyphagous grapevine pests (reviewed: [START_REF] Thiéry | Biological protection against grape berry moths. A review[END_REF]. These two species are responsible for significant damages in vineyards either directly by attacking grape bunchesa larva is able to damage between 2 and 10 berries depending on the grape cultivaror indirectly by favoring the development of several fungi, such as Botrytis cinerea, Aspergilus carbonarius, and Aspergilus niger [START_REF] Delbac | Damage to grape flowers and berries by Lobesia botrana larvae (Denis & Schiffernüller) (Lepidoptera: Tortricidae), and relation to larval age[END_REF][START_REF] Thiéry | Biological protection against grape berry moths. A review[END_REF]. Eupoecilia ambiguella mostly occurs in central Europe while L. botrana is widely distributed but mostly occurs at high density in Mediterranean climates [START_REF] Thiéry | Les ravageurs de la vigne[END_REF]. However, in several European vineyards, these two species can share common distributional range where they often live in sympatry, as in Beaujolais vineyards for example (North of Lyon, France) [START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF]. The density of each species within vineyards can vary from 15:75 to an equal distribution 50:50 (unpublished data). Despite such differences, biological control of E. ambiguella and L. botrana has always been the same, mostly performed through the release of the egg parasitoid Trichogramma sp. (Hymenoptera:

Trichogrammatidae) [START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF][START_REF] Thiéry | Biological protection against grape berry moths. A review[END_REF]. For the past decade, the larval parasitoid Campoplex capitator (Hymenoptera: Ichneumonidae) has drawn more and more attention (Marchesiniand and Dalla Monta 1994;[START_REF] Xuéreb | Does natural larval parasitism of Lobesia botrana vary between years, generation, density of the host and vine cultivar?[END_REF]. Cosmopolitan and very plastic in its ecological requirements [START_REF] Moreau | Species diversity of larval parasitoids of the European grapevine moth (Lobesia botrana, Lepidoptera: Tortricidae): The influence of region and cultivar[END_REF], this parasitoid is very efficient in regulating grapevine pest populations, with sometimes a level of parasitism rate up to 90% in L. botrana [START_REF] Vogelweith | Geographical variation in parasitism shapes larval immune function in a phytophagous insect[END_REF]. But, attempts to massing-rear C. capitor for the control of grapevine pests has always failed, chiefly because of the limited information about its biology/ecology, including the limited understanding of its ability to switch from the rearinghost (e.g. L. botrana) to hosts encountered in the field (e.g. L. botrana and E. ambiguella) (unpublished data). For instance, the larval-pupal parasitoid Psyttalia concolor (Hymenoptera: Braconidae) reared on the fruit fly Ceratitis capitata (Diptera: Tephritidae) (Canale and Benelli 2012) did not show any difference in oviposition behaviors and host acceptance when provided either with its rearing host C. capitata or the alternative host pest Bactrocera oleae (Diptera: Tephritidae) [START_REF] Canale | Impact of mass-rearing on the host seeking behaviour and parasitism by the fruit fly parasitoid Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae)[END_REF].

Here we seek to determine, for the first time, whether C. capitator females emerged from wild populations of L. botrana prefer E. ambiguella or L. botrana as host in a choice test, and consequently whether it could be a good candidate as bio-agent to regulate grapevine pest populations.

Material and methods

Model insects

Larvae of E. ambiguella and L. botrana used in this experiment came from two independent rearing facilities, maintained diapause-free at the French National Institute for Agricultural Research (INRA) (Villenave d'Ornon, France) for more than 15 years (see [START_REF] Vogelweith | No evidence of an immune adjustment in response to a parasitoid threat in Lobesia botrana larvae[END_REF]). These two strains were based on multiple adults reared in flying cages to which wild individuals were periodically added. Larvae of each species were provided with ad libitum semi-artificial diet [START_REF] Vogelweith | Food-mediated modulation of immunity in a phytophagous insect: An effect of nutrition rather than parasitic contamination[END_REF] and maintained in similar boxes (18 × 11.5 × 7 cm) under the same standard laboratory conditions (22 ± 1°C, 70 ± 10% RH, and a L16:D8 photoperiod) at a density of approximately 100 individuals per 300 ml of diet. Corrugated papers were placed within each box, allowing the larvae to hide, as they did in grape berries.

Female parasitoids of C. capitator were obtained from wild parasitized L. botrana larvae collected on Grenache cultivar in a French vineyard (N 42°447.063, E 2°5256.441; Perpignan, France) in May 2013 (see [START_REF] Vogelweith | Geographical variation in parasitism shapes larval immune function in a phytophagous insect[END_REF]. Sampled larvae were maintained in polyethylene boxes (60  40  21.4 cm) with ad libitum Grenache grape bunches from the vineyard, at 24 ± 1°C, 60 ± 10% RH and under the ambient photoperiod conditions. Corrugated papers were placed in the boxes to facilitate pupae collection (larvae like to shelter in such material before pupation) and checked daily for pupation. After pupation, pupae were individually moved into glass tubes (70 × 9 mm diameter) closed with cotton plugs under the conditions stated above and checked daily until emergence from either an adult moth or a parasitoid. Different parasitoid species emerged from larvae and after identification [START_REF] Thiéry | Les ravageurs de la vigne[END_REF], only C. capitator females (easily visually identifiable with their ovipositor) were kept for this experiment. Parasitoid females were transferred to clean glass tubes provided with a droplet of honey and cotton plugs soaked with water, but otherwise kept at the same conditions.

All C. capitator females used in this experiment were tested two days after their emergence, naïve (no prior oviposition) and were kept virgin because females of this species can also reproduce parthenogenetically, producing in such case only males [START_REF] Morris | Learning and natal host influence host preference, handling time and sex allocation behaviour in a pupal parasitoid[END_REF]Thiéry and Xuereb 2004).

Experimental design

A total of 22 C. capitator females were tested to determine their preference between E. ambiguella and L. botrana. Tests were carried out in an experimental arena constituted of a Petri dish (95 mm dimeter, 62 mm high) divided into two equal zones (Fig. 1): one zone dedicated to E. ambiguella, and the second dedicated to L. botrana. At the center of each zone was taped a piece of corrugated paper containing a 4 th instar larva of either E. ambiguella or L. botrana (Fig. 1). Prior to the test, all larvae were checked to ensure they were at the same instar (4 th instar) based on the larval head capsule size (1.2 ± 0.1 mm) [START_REF] Delbac | Larval instars determination for the European Grapevine Moth (Lepidoptera: Tortricidae) based on the frequency distribution of head-capsule widths[END_REF], and without physical damages. Then, larvae were moved to the small piece of corrugated paper using a paintbrush washed with 90% ethanol after each larva. The female parasitoid was then released in the middle of the experimental arena. Its behavior was recorded until the first attack (defined as the first oviposition attempt) or for a maximum of 30 min. During this time-period, the first species visited, the time spent by the parasitoid on each side of the arena (E. ambiguella versus L. botrana sides), the number of visits to each larva and the chosen larva (i.e. larva which suffered from an oviposition attempt) were recorded. As female C. capitator can perform multiple oviposition attempts in few seconds, inflicting serious injuries to the larva, a test was ended after the first attack. Each test was performed with a different female parasitoid and host larvae. The position of each species in the experimental arena was exchanged every time and a new arena was used for each repetition to avoid potential influence of cues/odor from both parasitoid and larvae. The experiment was performed for two hours (four individuals during 30 min per day), every day during six days, at the sunsettime-period where parasitoids are the most activeat 24 ± 1°C, 60 ± 10% RH and under the ambient photoperiod conditions.

Statistical analyses

All data analyses were conducted using the software R v3.5.1 loaded with car and lme4 packages. The proportion of time spend by the female parasitoid on each side was tested using a Chi-squared test for given probabilities where the proposition of time spent on the L. botrana side was compared to a probability of 0.5. The number of visits per host species was tested using a generalized linear model (GLM) with poisson distribution, and the species (L. botrana or E. ambiguella) entered as explanatory categorical factors. Finally, the first species visited as well as the choice made by the female parasitoid were tested using a generalized linear model (GLM) with binomial error distribution, and the species (L. botrana or E. ambiguella) entered as explanatory categorical factors.

Results

The first choice (i.e. the first larva visited) by C. capitator females was almost always L. botrana (95.45 % of the cases; F1 = 44.72; p < 0.0001). Moreover, females spent significantly more time on L. botrana side of the experimental arena (χ 2 1 = 7.68; p = 0.006; IC 95% = [0.59; 0.94]; Fig. 2a) and visited more often L. botrana larvae compared to E. ambiguella larvae (F1 = 10.11; p = 0.001; Fig. 2b). Finally, C. capitator females almost exclusively attacked L. botrana larvae (F1 = 31.18; p < 0.0001; Fig. 2c).

Discussion

Although host preference in parasitoids has been well studied, whether a parasitoid prefers its natal host or an alternative host in an agroecosystem hosting sympatric pest species remains unexplored. Based on a choice test experiment, our results clearly showed that C. capitator females emerged from wild population of L. botrana preferred the latter as host over E. ambiguella. Indeed, they went mostly directly to the L. botrana side, spent more time on this side and visited it more often compared to E. ambiguella larvae. Finally, L. botrana larvae were almost exclusively choose/attacked by C. capitator females.

At least three non-mutually exclusive hypotheses could explain such preference. The first hypothesis relies on the notion of NHPI [START_REF] Davis | The effect of natal experience on habitat preferences[END_REF], meaning that C. capitator females emerged from L. botrana prefer to lay their eggs in L. botrana larvae due to special stimuli received in early-life in their native habitat. Mounting evidence suggests the importance of the cues learned during ontogeny on parasitoid choice [START_REF] Morris | Learning and natal host influence host preference, handling time and sex allocation behaviour in a pupal parasitoid[END_REF][START_REF] Bodino | Is host selection influenced by natal and adult experience in the parasitoid Necremnus tutae (Hymenoptera: Eulophidae)?[END_REF][START_REF] Giunti | Early adult learning affects host preferences in the tephritid parasitoid Psyttalia concolor (Hymenoptera: Braconidae)[END_REF]. Such learning can take place after emergence or during pre-adult stages (when the parasitoid in still on/inside its host) and/or during adult emergence [START_REF] Giunti | Parasitoid learning: Current knowledge and implications for biological control[END_REF], as illustrated by the endoparasitoid P. concolor. Females from the latter excised from their host just before emergenceand consequently did not have contact with a natal host in early adult lifewere unable to show a preference for the natal host [START_REF] Giunti | Early adult learning affects host preferences in the tephritid parasitoid Psyttalia concolor (Hymenoptera: Braconidae)[END_REF]. Our results are consistent with the NHPI hypothesis even though we cannot determine whether C. capitator females are using pre-or post-emergence learning. The same experiment should be performed with E. ambiguella as natal host to confirm that statement. Parasitoid learning could also occur through an associative learning where they learn to associate cues from their environment to increase their foraging efficiency [START_REF] Hoedjes | Natural variation in learning rate and memory dynamics in parasitoid wasps: opportunities for converging ecology and neuroscience[END_REF]). In our study, parasitoid cocoons were removed from their environmental complex and placed under control conditions, meaning that associative learning was not tested here. As such, we do not know the consequences of host species choice upon C. capitator fitness. This point should be investigated in a further field survey where C. capitator live in an environment where L. botrana and E. ambiguella are in sympatry.

Secondly, parasitoids can also inherit natal environmental preferences related to a local adaptation that may result in a parasitoid population specializing on one host [START_REF] Gutiérrez-Ibáñez | Pre-pupation behaviour of the aphid parasitoid Aphidius ervi (Haliday) and its consequences for pre-imaginal learning[END_REF][START_REF] Zepeda-Paulo | Adaptive evolution of a generalist parasitoid: Implications for the effectiveness of biological control agents[END_REF][START_REF] Bodino | Is host selection influenced by natal and adult experience in the parasitoid Necremnus tutae (Hymenoptera: Eulophidae)?[END_REF]. A previous study suggested a local adaptation of L. botrana larvae to existing parasitoid pressure [START_REF] Vogelweith | Geographical variation in parasitism shapes larval immune function in a phytophagous insect[END_REF]). Since L. botrana and C. capitator co-evolved in vineyards for many years, it is also likely that C. capitator have adapted to local host species. Indeed, C. capitator used in this experiment came from an area where L. botrana are predominant compared to E. ambiguella [START_REF] Ricaud | Bilan de campagne 2013 viticulture en Provence Alpes Côte d'Azur et Sud Drôme Bull Santé du Végétal[END_REF].

Finally, the third hypothesis involves the defense strategies used by E. ambiguella and L. botrana larvae [START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF]. Despite many similarities in their ecological requirements and their natural enemies, these two species have been shown to invest differently in the defense mechanisms which might influence parasitoid choice [START_REF] Brodeur | The role of host species, age and defensive behaviour on ovipositional decisions in a solitary specialist and gregarious generalist parasitoid (Cotesia species)[END_REF][START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF]. Indeed, even though L. botrana larvae have been shown to express strong behavioral defenses, their cuticle and immune system are less resistant to parasitoids compared to E. ambiguella's [START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF]. Therefore, the choice performed by C. capitator is very consistent, and could be the result of a local adaptation of C. capitator to L. botrana larvae in the field, as mentioned in the second hypothesis.

In conclusion, these results show that biological control by augmentationrelying on the release the of natural enemies (i.e. C. capitator) that occur in the agroecosystemcould be very efficient against L. botrana populations due to the high specificity of its parasitoid. However, it might be less efficient against E. ambiguella populations, at least if C. capitator has L. botrana as natal host. More investigations about the relationship between C. capitator and E. ambiguella should be performed in order to consider this parasitoid as a potential bio-agent.

But, it is important to recall that studies and field reports indicated that C. capitator is parasitizing E. ambiguella in European vineyards [START_REF] Colombera | Comparison between the parasitoids of Lobesia botrana and Eupoecilia ambiguella in conventional and integrated vineyards[END_REF][START_REF] Thiéry | Control of grape berry moth larvae using parasitoids: should it be developed?[END_REF][START_REF] Thiéry | Biological protection against grape berry moths. A review[END_REF][START_REF] Rusch | Organic farming and host density affect parasitism rates of tortricid moths in vineyards[END_REF], even if parasitism success is higher in L. botrana than in E. ambiguella

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. (a) Percentage of time spend by the female C. capitator on E. ambiguella (E. a.) or

Figure 1

 1 Figure 1

Figure 2

 2 Figure 2

Acknowledgments

We thank Lionel Delbac for providing the larvae and Austin Alleman for proofreadings. We would also like to thank the editor and the two anonymous reviewers for their helpful comments. This study was supported by grants from the Conseil Regional of Bourgogne (J.M.).

when the ratio of both species in the population is close to 50:50 [START_REF] Vogelweith | Defense strategies used by two sympatric vineyard moth pests[END_REF].

Further investigations should be done in order to determine whether it would also be a good candidate to control E. ambiguella populations. Altogether, this study is one of the first to reveal C. capitator's potential as a valuable and efficient bio-agent for the control of grapevine moths in European vineyards.

Conflict of interest

The authors declare no competing financial interests.


Clotilde Biard

Karine Monceau

Maria Teixeira

Sébastien Motreuil

Soline Bettencourt-Amarante

Lucie Develay

Jérôme Moreau
email: jerome.moreau@u-bourgogne.fr

Soline Bettencourt- Amarante

Coccidial oocyst release: once a day or all day long? Tropical bird hosts shed new light on the adaptive significance of diurnal periodicity in parasite output

Keywords: circadian rhythms, Coccidia, temperate forest, tropical forest, host-parasite interactions, birds

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

Introduction

From an evolutionary perspective, natural selection should favour any trait increasing the probability of parasite transmission from one host to another [START_REF] Bush | Parasitism: the diversity and ecology of animal parasites[END_REF][START_REF] Combes | Parasitism: The ecology and evolution of intimate interactions[END_REF].

For example, many parasite species with complex life cycles (i.e., parasites with successive host species), have evolved strategies of releasing infective forms at times of the day when the definitive host is most likely to be present in the environment (Théron, 1984;[START_REF] Combes | Behaviours in trematode cercariae that enhance parasite transmission: patterns and processes[END_REF]Combes, 2001 and references therein). Such a phenomenon of diurnal periodicity is also observed in parasites with simple life cycles (i.e., parasites with a single host species). Coccidia from the genus Isospora (Protozoa, Apicomplexa) are intracellular intestinal parasites of birds, and typically peak oocyst release in the late afternoon [START_REF] Boughton | Diurnal gametic periodicity in avian Isospora[END_REF][START_REF] Kruszewicz | The occurrence of Isospora lacazei (Coccidia: Eimeriidae) and its influence on nestling growth in House Sparrows (Passer domesticus) and Tree Sparrows (Passer montanus)[END_REF][START_REF] Brawner | Temporal variation in shedding of coccidial oocysts: implications for sexual-selection studies[END_REF]Dolnik, 1999aDolnik, , 1999b;;[START_REF] Hudman | Effects of time of sampling on oocyst detection and effects of age and experimentally elevated testosterone on prevalence of coccidia in male dark-eyed juncos[END_REF][START_REF] Brown | The periodicity of isosporan oocyst discharge in the greenfinch (Carduelis chloris)[END_REF][START_REF] Misof | Diurnal cycle of Isospora spp. oocyst shedding in Eurasian blackbirds (Turdus merula)[END_REF]. These protozoans exhibit both sexual and asexual phases. In most species, one or more asexual multiplication's cycles are completed in the intestinal epithelium of the host, before sexual development occurs [START_REF] Olsen | Animal parasites: their life cycles and ecology[END_REF]. Zygotes produced during the sexual phase transform into oocysts that are then released into the host's intestinal tract and excreted within faeces. To complete their development, oocysts must sporulate outside the host to become infective before being ingested by their new host (see for an example Abd- [START_REF] Bettencourt- Amarante | Life-cycle of Isospora mehlhornii sp. nov. (Apicomplexa: Eimeriidae), parasite of the Egyptian swallow Hirundo rubicola savignii[END_REF].

In birds, coccidian oocyst shedding shows a strong circadian pattern that appears to be ubiquitous since in all species tested to date (16 species), oocyst shedding has been found to peak in the afternoon [START_REF] Knight | Chapter Two -The Evolutionary Biology, Ecology and Epidemiology of Coccidia of Passerine Birds[END_REF], whatever the latitude considered, from the tropics zone to artic area [START_REF] Dolnik | Keeping the clock set under the midnight sun: diurnal periodicity and synchrony of avian Isospora parasites cycle in the High Arctic[END_REF]. Even in an extreme environment with permanent daylight like the arctic, this periodicity is conserved despite the absence of diurnal periodicity in hosts' activity [START_REF] Dolnik | Keeping the clock set under the midnight sun: diurnal periodicity and synchrony of avian Isospora parasites cycle in the High Arctic[END_REF]. The peak in oocyst shedding late in the afternoon is considered an adaptative trait providing resistance to desiccation and UV radiations for oocysts in the external environment [START_REF] Martinaud | Circadian variation in shedding of the oocysts of Isospora turdi (Apicomplexa) in blackbirds (Turdus merula): An adaptative trait against desiccation and ultraviolet radiation[END_REF]. Indeed, a single hour of exposure of the host's faeces to natural sunlight reduced the viability of oocysts by ca. 50%, which further decreased with continued exposure [START_REF] Martinaud | Circadian variation in shedding of the oocysts of Isospora turdi (Apicomplexa) in blackbirds (Turdus merula): An adaptative trait against desiccation and ultraviolet radiation[END_REF]. Therefore, releasing oocysts throughout the day would result in a large proportion quickly dying due to sunlight exposure, and a decrease in transmission probability to the next host. Interestingly, all bird species showing temporal variation in the shedding of coccidia oocysts with a peak late in the afternoon roughly have in common one characteristic of their environment: a ground exposed to strong solar radiations including the arctic area [START_REF] Knight | Chapter Two -The Evolutionary Biology, Ecology and Epidemiology of Coccidia of Passerine Birds[END_REF]. However, an exciting question that remains open is whether oocysts shedding in an environment where the sun reaching the ground is dramatically reduced throughout the year still shows diurnal periodicity. Indeed, in such environments, the selective pressure of desiccation and UV exposure of oocysts during the daytime would be relieved. One would therefore predict periodicity in oocyst shedding either not to evolve or be lost as it would reduce transmission probability without the benefits of increasing oocysts viability and should thus be selected against.

Although tropical rainforests receive 12 hours of sunlight daily, it is generally assumed that less than 2% of radiations ever reach the ground. Tropical rainforests have dense vegetation, often forming three different layers including a dense canopy that blocks out ca. 95% of sunlight (Brinkman, 1971;[START_REF] Bourgeron | Spatial aspects of vegetation structure[END_REF][START_REF] Chazdon | Photosynthetic light environments in a lowland tropical rain forest in Costa Rica[END_REF]Vazquez-Yanes et al., 1990). Therefore, the rainforest ground rarely receives direct sunlight. Also, tropical rainforests are characterized by high air humidity, ranging on average from 80 to 90% relative humidity (RH; [START_REF] Freiberg | Spatial and temporal pattern of temperature and humidity of a tropical premontane rain forest tree in Costa Rica[END_REF][START_REF] Bruno | Soil moisture dynamics in an eastern Amazonian tropical forest[END_REF] and soil moisture. By combining low direct sunlight and high RH, this environment provides faeces shed by birds with very favourable conditions to allow oocyst sporulation and survival. If temporal variation in shedding of coccidial oocysts is under natural selection to avoid desiccation and UV radiations as hypothesized, we may predict that no circadian rhythm should be observed in tropical forests contrary to temperate forests. Indeed, temperate forests are exposed to a stronger irradiance and lower soil moisture compared to tropical forests since the vegetation canopy is moderately dense and allows light to penetrate [START_REF] Szwagrzyk | Dynamics of seedling banks in beech forest: results of a 10-year study on germination, growth and survival[END_REF]. However, to our knowledge, temporal variation in the shedding of coccidia oocysts has never been investigated in a tropical rain forest environment.

To investigate whether periodically synchronised release of coccidia oocysts from the genus Isospora may be absent in tropical forests, consistent with the hypothesis of relaxed natural selection linked to UV radiations and desiccation, we compared patterns of oocyst shedding from birds living in the primary rain forest in French Guyana (low luminosity and high humidity and moisture) and that from birds living in open temperate forests in mainland France (high ground luminosity and low humidity and moisture). The generality of this expected difference in patterns of oocyst shedding across the diversity in host ecology and life history was assessed by sampling a variety of bird species belonging to different families within both habitat types. We expected marked temporal variation in the shedding of coccidia oocysts for host species living in open forest and no or weak temporal variation for hosts living inside the dark rain forest. Besides, we took advantage of the spatial heterogeneity in vegetation cover existing around the field station located in the tropical rain forest to study variation in oocyst shedding patterns at a smaller scale. Indeed, a large area has been maintained open at the edge of the field station for more than 30 years (Fig. 1) and has selected bird species specialists of open habitats. We, therefore, expected temporal variation in the shedding of coccidia oocysts from host species inhabiting this open area, contrary to those inhabiting the close by rain forest.

Materials and methods

Trapping birds and sampling faeces

Adult males and females of different bird species (one-year-old and older, identified by plumage characteristics) were caught using mist-nets from sunrise to sunset in both tropical and temperate areas. Species and age were determined according to handbooks [START_REF] Hilty | Birds of Venezuela, 2 nd Edn[END_REF]Restall et al. 2006aRestall et al. , 2006b)). The full list of species and number of individuals caught is given in Table 1. In the temperate forest habitat, we sampled birds in a young deciduous forest located in Sologne (thereafter called Temperate Forest, France, 47°26'N, 2°13'E) during spring 2019 (10 sampling days). At that time of the season, sunrise occurs at 06:00 am and sunset around 09:00 pm. In the tropical forest, we sampled birds in the Amazonian primary rain forest during July and August 2018 and October 2019 (15 and 11 sampling days in total) in the two field sites of the Nouragues French research station: Saut-Pararé (thereafter Tropical Forest 1, French Guyana, 4°02'N, 52°41'W, 8 sampling days in 2018 and 11 sampling days in 2019) and Inselberg (thereafter Tropical Forest 2, French Guyana, 4°05'N, 52°41'W, 7 sampling days in 2018) where sunrise occurs at 06:00 am and sunset at 06:00 pm. In the first site, we sampled birds both within the forest undergrowth and at its edge in the open area close to the field camp (Fig. 1). On one typical sunny day, we measured abiotic parameters at ground level in these two areas (open and undergrowth): temperature (°C), UV index, luminosity intensity (in lux). These measures were performed 5 times over the course of the day (08:00 am, 10:00 am, 12:00 am, 15:00 pm, 17:30 pm), using a CROSCAAL phone -Trekker -X4 equipped with a software X4 sensors in 10 different spots randomly chosen to take spatial heterogeneity into account.

For birds that can be kept in captivity throughout their lives such as poultry, the preferred method to track temporal patterns of oocysts release as accurately as possible is to collect faeces every 2 h from sunrise to sunset for each bird maintained in an individual cage [START_REF] Filipiak | Caution on the assessment of intestinal parasitic load in studying parasite-mediated sexual selection: The case of blackbirds coccidiosis[END_REF]. However, in field conditions, enclosing wild birds in a cage for an entire day is not acceptable from an ethical point of view, due to the stress of capture and prolonged restraint, which may alter both their subsequent survival and ability to reproduce, as birds might have been breeding at the time of capture. To limit the stress imposed on birds, we followed the protocol adopted in snow bunting [START_REF] Dolnik | Keeping the clock set under the midnight sun: diurnal periodicity and synchrony of avian Isospora parasites cycle in the High Arctic[END_REF]. Birds were placed in individual cages, the bottom of which was lined with aluminium foil, at any time of capture during the day, for a maximum of one hour before being released at their site of capture, had the birds shed faeces or not. All fresh faeces present on the aluminium foil were collected and homogenised with a small spoon and stored at 4°C in 15 mL Falcon tubes labelled with the date, time of the day and bird species. To avoid repeated sampling of the same individuals, birds having shed faeces were marked with a drop of nontoxic nail varnish on the tarsus and in case of recapture, were not subjected again to faeces collection and were thus immediately released.

Prevalence and intensity of parasite infection by Isospora spp.

At the end of each field session in the two habitats (temperate/tropical), faeces samples were weighted to the nearest 0.01g using an electronic balance and a solution of Sheater (45% sugar in water) was added to a concentration of 1 mg of faeces for 4 ml of sheater, after which samples were gently homogenised. We sampled 600 µl of this solution on a McMaster chamber and allowed oocysts to float to the top for 10 min. All the oocysts of Isospora spp. under the grid of each chamber in the McMaster were counted using 10 x magnification. Two counts were obtained for each individual faecal sample, and the average concentration, expressed as the number of oocysts per gram of faecal sample, was used as an estimate of oocyst production rate.

Statistical analyses

The time of faeces collection was first calculated for each bird as the average hour between entrance and release from the cage. This time was standardized in the proportion of daily sunshine duration to take differences in day length between tropical and temperate sites into Differences in prevalence and parasite load between temperate (Temperate Forest) and tropical environments (Tropical Forest 1 + Tropical Forest 2) in birds sampled in the forest (birds caught in the open area in Tropical Forest 1 were excluded from this analysis) were analyzed using a Generalized Linear Mixed-Effects Model with Binomial distribution (B-GLMM, using glmer function) and Negative Binomial distribution (NB-GLMM, using glmmTMB function) respectively, including either the standardized hour or part of the day, habitat (temperate vs. tropical) and their interaction as fixed effects. Species identity was included as a random effect. The slope of fitted lines/curves were provided assorted with their 95% confidence interval from the models. Similar models were run to compare the two sites in the tropical habitat (i.e. Tropical Forest 1 vs Tropical Forest 2).

The same procedures were used to investigate differences in prevalence and parasite load in birds from open area versus surrounding undergrowth in the Tropical Forest 1.

Differences in abiotic factors during the day (temperature, UV radiations and luminosity) between these two habitats were analyzed including the standardized hour of the day as quadratic effect to test for a potential maximal effect at midday and the location and their interaction. The temperature was analyzed using a Gaussian GLM (glm function) while UV radiation and luminosity were analyzed using NB-GLM (glm.nb function).

For all GLMs and GLMMs, model comparisons were made using a deviance analysis for unbalanced design based on the likelihood ratio χ²-statistics or F-ratio for Gaussian GLM to test the contribution of the variable. Interactions were discarded when non-significant.

Results

Differences in temporal patterns of parasite prevalence in species from Temperate and Tropical

Forests

The prevalence of Isospora spp. oocysts in bird faeces increased dramatically as the day progressed for species inhabiting temperate forest to reach a maximum of 90% of infected individuals (slopeTemperate-Forest [95%CI] = 6.89 [5.27; 8.50]) whereas in the same time the prevalence for species living in tropical forest remained unchanged around 25% of infected individuals (slopeTropical-Forest [95%CI] = 0.86 [-0.40; 2.12]) (B-GLMM: effect of time: χ²1= 69.76, P < 0.0001, effect of location: χ²1= 6.65, P < 0.01, effect of their interaction: χ²1= 33.72, P < 0.0001)(Fig. 2). The prevalence of Isospora spp. oocysts in bird faeces did not differ between the two tropical forest sites (B-GLMM: effect of time: χ²1= 2.47, P = 0.12, effect of sites: χ²1= 1.70, P = 0.19, effect of their interaction: χ²1= 0.98, P = 0.32, slopeTropical-Forest1[95%CI] = 0.43 [-1.18; 2.05] and slopeTropical-Forest2 [95%CI] = 1.87 [-0.42; 4.20]).

Complementary analyses of parasite prevalence contrasting morning and afternoon data are presented in Table 1 and 222.81 ± 46.01 oocysts per bird). In contrast, parasite load increased over time from 0 to around 200 oocyst per bird at sunset (slopeTropical-Forest [95%CI] = 5.98 [2.27; 9.69], mean ± s.e. parasite load: 80.72 ± 30.97 oocysts per bird), (NB-GLMM: effect of time: χ²1= 0.00, P = 1.00, effect of location: χ²1= 7.86, P < 0.01, effect of their interaction: χ²1= 5.84, P < 0.05) (Fig. 3). In the Tropical Forest, the number of oocyst released in bird faeces increased through the afternoon and did not differ between the two sites and (NB-GLMM: effect of time: χ²1= 6.94, P < 0.01, effect of sites: χ²1= 0.14, P = 0.71, effect of their interaction: χ²1= 0.02, P = 0.89, slopeTropical-Forest 1 [95%CI] = 6.18 [1.73; 10.6] and slopeTropical-Forest 2 [95%CI] = 6. 64 [1.36; 11.9]).

Differences in temporal patterns of prevalence and intensity between open field and undergrowth in the Tropical Forest 1

Prevalence of Isospora infection increased in the open area but remain stable inside the Tropical Forest throughout the day (B-GLMM: effect of time: χ²1= 4.90, P < 0.05; effect of area: χ²1= 2.66, P = 0.10, effect of their interaction: χ²1= 4.30, P < 0.05; slopeopenarea [95%CI] = 8.87 [1.02; 16.72] versus slopeundergrowth [95%CI] = 0.47 [-1.09; 2.02])(Fig. 4). For example, in the most abundant species captured in this open area (Silver-beaked Tanager), all individuals caught in the afternoon shed oocyst in their faeces whereas no individual caught in the morning did (Table 1). Considering only infected birds in the afternoon (standardized hour > 0.5), parasite load was higher in the open area than in the undergrowth (NB-GLMM: effect of time: χ²1 = 1.08, P = 0.30; effect of area: χ²1 = 5.35, P < 0.05, mean ± s.e. parasite load open area: 1028.70 ± 441.20 oocysts/bird and forest undergrowth: 91.06 ± 44.13 oocysts/bird) (Fig. 5).

Comparison of abiotic factors between the forest undergrowth and open area

Variation in temperature showed a curvilinear relationship with time elapsed since sunrise, with a peak around 35°C in open area and only around 30°C in the forest undergrowth between 12:00 and 15:00 (Gaussian GLM: time x area: F1,94 = 4.88, P = 0.03, time 2 x area: F1,94 = 5.20, P = 0.02, Fig. 6a). In open area, UV radiation and luminosity showed a similar relationship with time from the morning to midday to reach a high intensity of UV radiation and luminosity intensity before a strong decrease until the sunset. In contrast, in the forest undergrowth UV radiation and luminosity intensity remains at very low level throughout the day (NB-GLM for UV radiation: time: χ²1 = 56.39, P < 0.0001; time 2 : χ²1 = 59,99, P < 0.0001; location: χ²1 = 169.08, P < 0.0001, Fig. 6b and NB-GLM for luminosity: time: χ²1 = 52.01, P < 0.0001; time 2 : χ²1 = 57.30, P < 0.0001; location: χ²1 = 188.73, P < 0.0001, Fig. 6c).

Discussion

In this study, we expected circadian variation in coccidial oocysts shedding to be found in temperate, but not in tropical forest habitat, consistent with the hypothesis that this trait in parasites is maintained by natural selection to avoid desiccation and UV radiation, to which oocysts are sensitive before sporulation. We predicted that in an environment where solar radiations reaching the ground are dramatically reduced, like in the dense undergrowth of a tropical forest, the circadian output of oocysts typically found in bird coccidia would disappear.

Our results clearly confirmed this prediction since circadian variation in coccidian oocysts was high in the temperate forest (i.e., 90 % of bird were infected in the late afternoon and almost none in the morning) and absent in the tropical forest (approximatively 20% of bird's sheds oocyst in their faces whatever the hour of trapping). Furthermore, at a smaller spatial scale in the Tropical Forest 1 site, a similar result was found, as birds captured in the open area showed a strong circadian variation contrary to bird species from the surrounding undergrowth of the rain forest.

The main goal of this study was to go one step further regarding the adaptive significance of circadian variation in coccidial oocysts shedding in birds, demonstrated in all tested species including birds living in the arctic area [START_REF] Knight | Chapter Two -The Evolutionary Biology, Ecology and Epidemiology of Coccidia of Passerine Birds[END_REF] for a review). Ten years earlier, [START_REF] Martinaud | Circadian variation in shedding of the oocysts of Isospora turdi (Apicomplexa) in blackbirds (Turdus merula): An adaptative trait against desiccation and ultraviolet radiation[END_REF] experimentally demonstrated that the release of oocysts in the late afternoon was an adaptative trait to avoid desiccation and UV radiation, thus reducing mortality of oocysts in the external environment. Before our study, such a circadian pattern appeared to be a ubiquitous phenomenon in intestinal bird parasites, since all studies showed that oocyst shedding peaks in the late afternoon (reviewed in [START_REF] Knight | Chapter Two -The Evolutionary Biology, Ecology and Epidemiology of Coccidia of Passerine Birds[END_REF]). To our knowledge, this study is the first to document the absence of circadian variation in coccidial oocysts shedding in a large set of species inhabiting the undergrowth of a rain forest. This environment is characterized by low luminosity and low UV radiation levels reaching the ground, combined with high soil and air moisture [START_REF] Chazdon | Photosynthetic light environments in a lowland tropical rain forest in Costa Rica[END_REF]Vazquez-Yanes et al., 1990). All these abiotic characteristics are favourable for the survival of oocysts in host faeces [START_REF] Allen | Recent advances in biology and immunobiology of Eimeria species and in diagnosis and control of infection with these coccidian parasites of poultry[END_REF]. If temporal variation in the shedding of coccidial oocysts is a trait that has evolved under a natural selection to avoid desiccation and UV radiation as commonly thought [START_REF] Martinaud | Circadian variation in shedding of the oocysts of Isospora turdi (Apicomplexa) in blackbirds (Turdus merula): An adaptative trait against desiccation and ultraviolet radiation[END_REF], such a pattern should not be observed in an environment where these abiotic selective pressures are relaxed. Contrary to the continuous multi-layered vegetation canopy of rain forests, the canopy in temperate forests is moderately dense and allows greater and seasonally varying light penetration [START_REF] Anderson | Studies of the woodland light climate: II. Seasonal variation in the light climate[END_REF][START_REF] Koizumi | Seasonal changes in photosynthesis of four understory herbs in deciduous forests[END_REF][START_REF] Szwagrzyk | Dynamics of seedling banks in beech forest: results of a 10-year study on germination, growth and survival[END_REF]. Moreover, most temperate forests are managed and affected by human activities, and light intensities reaching the ground may differ greatly according to the stage in the forest management [START_REF] Canham | The response of woody plants to disturbance: patterns of establishment and growth[END_REF][START_REF] Buckley | Ecology and management of coppice woodlands[END_REF][START_REF] Mcelrone | Water uptake and transport in vascular plants[END_REF]. For example, in most recent cuts (1 year-old), light intensity was 3.5 to 5 times higher than in uncut plots, and rapidly decreased over time following selection cutting, especially at ground level [START_REF] Beaudet | Understorey light profiles in temperate deciduous forests: recovery process following selection cutting[END_REF]. Therefore, temperate forests are exposed to a stronger and more variable irradiance compared to tropical rain forest. We observed marked temporal variation in the shedding of coccidial oocysts in birds from the temperate forest, consistent with previous studies [START_REF] Knight | Chapter Two -The Evolutionary Biology, Ecology and Epidemiology of Coccidia of Passerine Birds[END_REF]. On the contrary, bird species living in the tropical forest did not show this diurnal variation. These results suggest that natural selection (evolutionary costs and benefits) may indeed shape release rhythms in Isospora spp. of birds like in many other parasite species [START_REF] Reece | The life and times of parasites: Rhythms in strategies for within-host survival and between-host transmission[END_REF]. in the open area were on average 100 times higher compared to the ground in the undergrowth, at any time but in particular at midday. Furthermore, the field camp is located near the equator, a place where the annual UV doses reach their maximum [START_REF] Marks | The environmental threat to the skin[END_REF], increasing the selective pressure towards a marked circadian variation in coccidial oocysts shedding.

Therefore, Isopora sp. infecting birds living in this open area have likely been selected to evolve strong temporal variation in oocysts shedding allowing oocyst survival in such a harsh external environment.

Regarding variation in the intensity of Isospora spp. infection, we found that parasite load remained stable in temperate species through the day while it increased in tropical areas, to reach by the end of the afternoon approximately the same parasite load as in temperate species. This increase in oocyst release during the day confirms previous results on two species (Serinus serinus and Sylvia borin) in which coccidial parasite load in the faeces peaked in the late afternoon [START_REF] Lopez | Time of day, age and feeding habits influence coccidian oocyst shedding in wild passerines[END_REF]. The mechanisms underlying this process, however, are still unclear. After different phases of multiplication inside the intestinal tractus of its host, oocysts are released from its host cell and shed into the environment with the faeces. The proximal factors triggering coccidian oocyst discharge are not identified yet but it is known that they are linked to host physiology [START_REF] Boughton | Diurnal gametic periodicity in avian Isospora[END_REF]Dolnik, 1999aDolnik, , 1999b) ) and potentially mediated by melatonin, which regulates daily activity patterns in vertebrates. Indeed, outside polar regions with constant daylight, avian Isospora species use cyclic changes in melatonin concentration in their host blood to synchronize oocyst output (Dolnik et al. 2011, and references therein).

Therefore, we may hypothesize that the threshold of physiological signals inducing oocyst discharge is very low for tropical species inhabiting the dense undergrowth, allowing oocysts discharge early in the morning. The accumulation of these signals as the day progressed may induce the slight but continuous increase in parasite load observed in our data over the afternoon. Another but not exclusive hypothesis would be that the amount of faeces produced could be higher in the late afternoon allowing a higher discharge in oocyst in the same time.

Finally, our results meet an interesting methodology issue for researchers interested in working on intestinal parasites like Isospora spp. in wild birds' species [START_REF] Biard | Interpreting immunological indices: The importance of taking parasite community into account. An example in blackbirds Turdus merula[END_REF][START_REF] Biard | Carotenoid-based bill colour is an integrative signal of multiple parasite infection in blackbird[END_REF]. Due to circadian rhythm in oocyst shedding in temperate habitat there is a large difference in the number of Isopora sp. oocysts shed between the morning and afternoon for a given individual [START_REF] Filipiak | Caution on the assessment of intestinal parasitic load in studying parasite-mediated sexual selection: The case of blackbirds coccidiosis[END_REF]. Hence, to accurately assess, and make meaningful comparisons in, the prevalence of Isospora infection, sampling of faeces must be standardized at the same time of the day, as already demonstrated in previous studies [START_REF] Filipiak | Caution on the assessment of intestinal parasitic load in studying parasite-mediated sexual selection: The case of blackbirds coccidiosis[END_REF][START_REF] Dolnik | The effect of host foraging ecology on the prevalence and intensity of coccidian infection in wild passerine birds[END_REF]. In the tropical rain forest where this circadian rhythm in oocyst shedding is absent, prevalence of infection may be assessed from faeces collected at any time of the day. This makes it easier to work on the effects of intestinal parasites on bird host life-history traits such as sexually selected traits for instance [START_REF] Brawner | Effects of coccidial and mycoplasmal infections on carotenoid-based plumage pigmentation in male House finches[END_REF][START_REF] Hõrak | How coccidian parasites affect health and appearance of greenfinches[END_REF]. However, as parasite load increases as the day progresses, we emphasise the need to precisely record the time of day at which samples are collected. If Isospora spp. parasite loads are to be compared, then sampling time should be standardized at the same time of day.

Concluding remarks

We believe that studying tropical birds improved our knowledge of the ecology of Isospora spp. in natural populations of hosts and the adaptive significance of daily rhythm in oocyst shedding. The available information on intestinal parasites in tropical birds to date is scarce as research has often focused on blood parasites [START_REF] Ricklefs | Embryonic development period and the prevalence of avian blood parasites[END_REF][START_REF] Durrant | Avian hematozoa in South America: A comparison of temperate and tropical zones[END_REF][START_REF] Ricklefs | Malaria prevalence and white-blood-cell response to infection in a tropical and in a temperate thrush[END_REF][START_REF] Merino | Haematozoa in forest birds from southern Chile: Latitudinal gradients in prevalence and parasite lineage richness[END_REF][START_REF] Svensson-Coelho | Reciprocal specialization in multihost malaria parasite communities of birds: a temperate-tropical comparison[END_REF]. Our study clearly showed that the knowledge on the interaction between parasite and host in the temperate area cannot be transposed for birds in tropical area. Therefore, future investigations are needed to reveal more information's on host-parasite interactions concerning the probability to be infected in the two different ecosystems. Comparative analysis with a large set of species would be very useful to investigate the ecological factors and host life-history traits underlying parasite diversity across different ecological contexts (tropical versus temperate).

Supplementary material

No supplementary material

  account as follow : ℎ𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑𝑖𝑧𝑒𝑑 = ℎ𝑎𝑣𝑒𝑟𝑎𝑔𝑒-ℎ𝑠𝑢𝑛𝑟𝑖𝑠𝑒 ℎ𝑠𝑢𝑛𝑠𝑒𝑡-ℎ𝑠𝑢𝑛𝑟𝑖𝑠𝑒 with haverage the average hour of collection, hsunrise and hsunset the sunrise and sunset time. Additionally, to compare morning and afternoon prevalence and infection intensity, daylength was divided into two parts: morning: standardized hour < 0.5 and afternoon: standardized hour > 0.5. All statistical analyses were performed with R software (v.4.0.4, R Core Team 2021) implemented with packages MASS, car for model comparison for unbalanced design and emmeans for post hoc pairwise comparison tests.

  yielded the same results. Differences in temporal patterns of infection intensity between species of Temperate and Tropical Forest Pre-analyses showed differences in infection intensity, measured by parasite load in the host faeces, between morning and afternoon in Temperate Forest (Wilcoxon test: W = 983.5, P = 0.004), and in Tropical Forest 1 (Wilcoxon test: W = 206.5, P = 0.01) but not in Tropical Forest 2 (Wilcoxon test: W = 29.5, P = 0.38)(mean ± s.e. of number of Isospora spp. shed in faeces in the morning versus in the afternoon respectively: Temperate Forest: 35.36 ± 15.95 versus 222.81 ± 46.01, Tropical Forest 1: 29.71 ± 24.88 versus 91.06 ± 44.13 and Tropical Forest 2: 27.20 ± 23.25 versus 62.33 ± 37.92). Therefore, analyses of differences in parasite load were restricted to data from infected birds sampled in the afternoon (excluding birds from the open area in Tropical Forest 1). Parasite load remained constant all the afternoon in species living in temperate habitat (slopeTemperate-Forest [95%CI] = -0.002 [-3.01; 3.01]; mean ± s.e. parasite load:

  In addition, comparing circadian variation in coccidial oocysts shedding within the tropical habitat between species exclusively living in the open area and those living in the surrounding undergrowth further support this hypothesis. This open area was created at the construction of the field camp, more than 30 years ago, and has since selected bird species specialized in open habitats. Indeed, species captured in the open field were never caught in the undergrowth, like Silver-beaked Tanager (Ramphocelus carbo carbo), Golden-sided Euphonia (Euphonia cayennensis), Violaceous Euphonia (Euphonia violacea), Tropical Kingbird (Tyrannus melancholicus), Great Kiskadee (Pitangus sulphuratus), Buff throated saltator (Saltator maximus) and Ruffous browed peppershike (Cyclarhis gujanensis). A review of the literature (Handbook of the Birds of the World Series) confirms that these bird species are specialists of open habitats. Unlike the surrounding undergrowth, this open area is characterized by strong level of luminosity and UV radiation reaching the ground. Indeed, during a typical day in the tropical environment, light intensity and UV radiation reaching the ground measured

Figure 1 :Figure 2 :

 12 Figure 1: Location of the open area inside the Tropical Forest 1 -French Guyana, Nouragues

Figure 3 :

 3 Figure 3: Parasite load of Isopora spp. (number of oocysts per gram of faeces) in different bird

Figure 4 :

 4 Figure 4: Prevalence of Isospora spp. (probability of infection, 1: infected, 0: not infected) in

Figure 5 .

 5 Figure 5. Parasite load of Isopora spp (number of oocysts per gram of faeces) in different bird

Table 2 :

 2 Post hoc tests based on the B-GLMM for the comparisons of morning (AM) and afternoon (PM) prevalence in temperate and tropical areas (corrected for multiple comparisons, see text for details). Significant differences are in bold.

	Contrasts	estimate	Standard error Z ratio	P
	temperate PM -AM	3.70	0.42	8.73	< 0.0001
	tropical PM -AM	0.37	0.34	1.10	0.32
	temperate AM -tropical AM	-0.27	0.42	-0.63	0.53
	temperate PM -tropical PM	3.06	0.41	7.45	< 0.0001
	temperate PM -tropical AM	3.43	0.42	8.23	< 0.0001
	tropical PM -temperate AM	0.64	0.42	1.51	0.20

Acknowledgements

This work has been conducted within the French natural reserve of Nouragues. We thank the Nouragues CNRS research field station (USR mixte LEEISA, CNRS; Cayenne) and the staff dedicated. We would like to thank all the people who contributed to the fieldwork.

Data

Once the paper has been accepted, data will be deposited in the Dryad

Author Contribution

CB, KM and JM conceived and designed the study. CB, MT, SM, SBA, LD and JM conducted data gathering. KM and JM performed statistical analyses. CB, KM and JM wrote the article.

Financial Support

We thank the Nouragues CNRS research field station, which benefits from "Investissement d'Avenir" grants managed by Agence Nationale de la Recherche (AnaEE France ANR-11-INBS-0001; Labex CEBA ANR-10-LABX-25-01).

Conflicts of Interest

The authors declare there are no conflicts of interest.

Ethical Standards

This work conforms to French legal requirements, and accepted international ethical standards, including those relating to conservation and welfare, and to the journal's policy on these matters. We thank the Direction Régionale de l'Environnement, de l'Aménagement et du Logement (DREAL) de la Guyane, de la Nouvelle-Aquitaine et du Loir et Cher for delivering legal authorizations of catching birds (authorizations no: R03-2018-07-17-002, R03-2019-03-14-005, 128/2017, 2019D/2323, 41-2018-04-03-002).

Théron, A (1984) separately for birds caught in the morning (h standardized < 0.5) and in the afternoon (h standardized > 0.5). Birds were caught in three different environments: Temperate Forest (Souesmes), Tropical Forest 1 (Nouragues, Pararé) undergrowth and open area, Tropical Forest 2 (Nouragues, Inselberg). Differences in parasite prevalence in host faeces between morning and afternoon differed between tropical and temperate species (B-GLMM: effect of morning vs. afternoon: χ²1= 76.21, P < 0.0001, effect of the site: χ²1= 55.41, P < 0.0001, effect of their interaction: χ²1= 38.03, P < 0.0001). In the Temperate Forest, the prevalence of Isospora was higher in the afternoon than in the morning, whereas prevalence did not differ between morning and afternoon in the Tropical Forest (


Aurélie Antunes

Lucie Jaquillard

Evelyne Benoit

Denis Servent

Aurélien Claeyssen

EAE induction using MOG35-55 : impact of CFA amount

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

Introduction

Experimental autoimmune encephalomyelitis (EAE) is one of the most popular animal models of multiple sclerosis (MS). Mouse models of demyelinating diseases have been useful in both the demonstration of T cellmediated demyelination and in the characterization of the pathogenesis of immune-mediated demyelinating disease. EAE is a CD4 + T cell-mediated, demyelinating autoimmune disease of the CNS that is characterized by mononuclear cell infiltration. EAE can be induced by various antigens including MOG35-55, a peptide derived from the Myelin Oligodendrocyte Glycoprotein, a structural protein in the myelin sheath. The injection of this peptide emulsified with CFA in mice induces a production of anti-MOG antibodies that cause demyelination and a chronic EAE. The purpose of this study was to investigate the influence of CFA amount on EAE induction.

Materials & methods

For this experience twenty C57BL/6 mice were acclimatized for 12 days before immunization on Day 0. They have been treated in strict adherence with the European Community guidelines for laboratory animal handling and with the guidelines established by the French Council on animal care "Guide for the Care and Use of Laboratory Animals". They had a free access to water and food and a room with controlled temperature and a 12h light/12h darkness cycle.

For the immunization the mice were divided in 4 experimental groups. A volume of 200µL with below emulsion preparations were injected at Day 0 in s.c.. The pertussis toxin (300ng/kg) was injected at Days 0 and 2 in i.p.

Emulsions were prepared by SB-PEPTIDE (catalog reference SB036, « premade EAE induction kit ») using mouse MOG35-55 (catalog reference SB023).

To classify clinical signs during the progression of EAE, observations have been made every day during 6 weeks using the clinical score indicated below.

Results

The results show that the mean clinical score of the three tested groups of mice started to increase, 14 days after immunization, from 0.0 to a maximum value over 2.0, while it remained null for all the animals of the G4 control group. After Day 20, the G3 mice have seen their clinical score to increase to 3.0 for the next 20 days.

Conclusion

These results indicate that the EAE progression appears to occur more rapidly and more potently when the CFA amount was increased.

Application note

  No symptoms 0.5 -Partial paralysis of the tail 1.0 -Total paralysis of the tail 1.5 -Dragging the hip, but fully moving the hindlimb 2.0 -Partial paralysis of the two hindlimbs 2.5 -Total paralysis of one hindlimb and partial of the second limb 3.0 -Total paralysis of the two hindlimbs 3.5 -Total paralysis of the two hindlimbs and column arched, but still moving the forelegs 4.0 -Total paralysis of the hindlimbs and partial paralysis of forelimbs (end-point) 5.0 -Decreased responsiveness (end-point) and death Discover SB-PEPTIDE's EAE induction products -peptide.com -contact@sb-peptide.com Antunes A. 1,2 , Jaquillard L. 1 , Benoit E. 2 , Servent D. 2 , Claeyssen A. 1 1 SB-PEPTIDE, 6 rue des platanes, 38120 Saint Egrève, France 2 SIMoS -CEA de Saclay -Gif sur Yvette


Jonas Ogien

Arnaud Dubois

Compact LED-based full-field optical coherence microscopy for high-resolution high-speed in vivo imaging

Keywords: Optical coherence tomography, medical and biological imaging, interference microscopy

This work reports on a compact full-field optical coherence microscopy (FF-OCM) setup specifically designed to meet the needs for in vivo imaging, illuminated by a high-brightness broadband light emitting diode (LED). Broadband LEDs have spectra potentially large enough to provide imaging spatial resolutions similar to those reached using conventional halogen lamps, but their radiance can be much higher, which leads to high speed acquisition and makes in vivo imaging possible. We introduce a FF-OCM setup using a 2.3 W broadband LED, with an interferometer designed to be as compact as possible in order to provide the basis for a portable system that will make it possible to fully benefit from the capacity for in vivo imaging by providing the ability to image any region of interest in real-time. The interferometer part of the compact FF-OCM setup weighs 210 g for a size of 11115 cm 3 . Using this setup, a sub-micron axial resolution was reached, with a detection sensitivity of 68 dB at an imaging rate of 250 Hz. Due to the high imaging rate, the sensitivity could be improved by accumulation while maintaining an acquisition time short enough for in vivo imaging. It was possible to reach a sensitivity of 75 dB at a 50 Hz imaging rate. High resolution in vivo human skin images were obtained with this setup and compared with images of excised human skin, showing high similarity.

INTRODUCTION

Optical Coherence Tomography (OCT) is a low coherence interferometric imaging technique capable of vertical crosssectional imaging of biological tissues with micrometer scale resolution in a non invasive way [START_REF] Huang | Optical coherence tomography[END_REF] . It has initially drawn much attention in the field of ophthalmology [START_REF] Swanson | In vivo retinal imaging by optical coherence tomography[END_REF] where it has been established as the gold standard for retinal imaging in a remarkably short amount of time, as it constitutes a powerful tool for monitoring retinal layers morphology in real-time, in vivo. OCT has also seen much interest in the field of dermatology [START_REF] Welzel | Optical coherence tomography in dermatology: a review[END_REF] , nevertheless, in conventional frequency domain (FD) OCT, the lateral resolution is limited by the depth of focus of the system, which must be large enough to match at least the penetration depth as there is conventionally no refocusing process. This constitutes a limitation for dermatological imaging were cellular-level imaging is often of importance.

Full-Field Optical Coherence Microscopy (FF-OCM), also referred to as Full-Field OCT (FF-OCT) is a parallelized approach of OCT where en face images are recorded using an area sensor and full-field illumination [START_REF] Beaurepaire | Full-field optical coherence microscopy[END_REF][START_REF] Vabre | Thermal-light full-field optical coherence tomography[END_REF][START_REF] Dubois | High-resolution full-field optical coherence tomography with a Linnik microscope[END_REF][START_REF] Dubois | Ultrahigh-resolution fullfield optical coherence tomography[END_REF][START_REF] Oh | Ultrahigh-resolution fullfield optical coherence microscopy using InGaAs camera[END_REF][START_REF] Dubois | Thermal-light full-field optical coherence tomography in the 1.2m wavelength region[END_REF][START_REF] Dubois | Handbook of full-field optical coherence microscopy: technology and applications[END_REF] . In en face imaging, there is no depth of field constraint as the image is obtained in a given plane, and therefore microscope objectives of higher numerical apertures (NA) can be used to achieve higher transverse resolution. Cellular level imaging within the skin is therefore achievable and FF-OCM has seen developments oriented towards dermatological imaging [START_REF] Dalimier | Full-Field Optical Coherence Tomography: A New Technology for 3D High-Resolution Skin Imaging[END_REF][START_REF] Tsai | Full-depth epidermis tomography using a Mirau-based full-field optical coherence tomography[END_REF] . Nevertheless, contrary to FD-OCT where acquisition of the interferometric data is very fast due to the development of high-speed swept-source lasers, FF-OCM speed is generally a limiting factor regarding in vivo imaging. Up to now, there have been several efforts to make in vivo imaging feasible with FF-OCM by improving imaging speed. Implementations of in vivo FF-OCM included the use of single-shot setups using two cameras, or the use of high brightness light sources in order to yield fast acquisition of the interferometric data, and sometimes a combination of those two methods [START_REF] Akiba | In vivo video-rate cellular-level full-field optical coherence tomography[END_REF][START_REF] Subhash | Full-Field and Single-Shot Full-Field Optical Coherence Tomography: A Novel Technique for Biomedical Imaging Applications[END_REF][START_REF] Latrive | In vivo and in situ cellular imaging full-field optical coherence tomography with a rigid endoscopic probe[END_REF][START_REF] Grieve | In vivo anterior segment imaging in the rat eye with high speed white light full-field optical coherence tomography[END_REF][START_REF] Moneron | Stroboscopic ultrahigh-resolution full-field optical coherence tomography[END_REF][START_REF] Dunsby | Single-shot phase stepped wide-field coherence gated imaging[END_REF][START_REF] Hrebesh | In vivo imaging of dynamic biological specimen by real-time single-shot full-field optical coherence tomography[END_REF][START_REF] Sacchet | Motion artifact suppression in fullfield optical coherence tomography[END_REF][START_REF] Schausberger | Full-field optical coherence microscopy with Riesz transform-based demodulation for dynamic imaging[END_REF] . Nevertheless, owing to the fact that some of those implementations have been done at the price of a loss of image quality and that most of them are complex and/or expensive, in vivo imaging is still not a common feature of FF-OCM. *jonas.ogien@institutoptique.fr; phone +331. 64.53.32.71 This paper introduces a simple and inexpensive FF-OCM setup designed for in vivo imaging, based upon a Linnik interference microscope and using a single camera. A high-brightness broadband light emitting diode (LED) is employed as the light source for high speed and high spatial resolution imaging. Furthermore, an innovative and compact design is introduced in order to provide the basis for a portable system which will properly meet the needs for in vivo imaging, that is to easily image any region of interest in real-time. The setup is characterized in terms of axial and transverse resolutions, detection sensitivity and operation speed. Application of the setup for biomedical imaging is then investigated by imaging human skin in vivo and ex vivo.

HIGH-SPEED IMAGING WITH FF-OCM

The imaging speed in FF-OCM is mainly determined by several factors: radiance of the light source, full well capacity (FWC) of the camera sensor, and number of interferometric images needed in order to reconstruct an en face tomographic image.

Radiance and FWC

In FF-OCM, in order to obtain the best possible imaging sensitivity for a given camera sensor, it is necessary that its pixels FWC is almost completely filled during image acquisition (i.e. the pixels are close to saturation). As sensitivity is a critical parameter, cameras with large FWC (i.e high dynamic cameras) should be used in FF-OCM for biomedical imaging, in order to reach sensitivities high enough to image very low reflecting features within biological tissues. Therefore, the time required to record one image (the integration time of the camera) with filled FWC is directly linked to the irradiance upon the camera sensor. The higher it is, the faster the FWC is filled, and the faster imaging is, with no loss regarding sensitivity. Irradiance upon the camera sensor being linked to the radiance of the light source according to Eq. ( 1) considering a simple approximation where the radiance is uniform, with E being the irradiance in the sensor plane, K being the percentage of the spectrum of emission of the light source actually detected by the sensor (considering its quantum efficiency, and losses within the optical system), L being the radiance of the light source, G being the limiting geometrical extent of the optical system, and A being the area illuminated over the sensor plane (considered uniform as the radiance is uniform):

E = KG A L (1)
For a given FF-OCM system, the higher the radiance of the light source, the faster the system can acquire interferometric data. Note that it is often difficult to reach very low integration times in FF-OCM as, by principle, the whole field to be imaged is illuminated, meaning that A takes rather large values in Eq. (1), and spatially incoherent light sources must be used in order to avoid artifacts [START_REF] Karamata | Spatially incoherent illumination as a mechanism for cross-talk suppression in wide-field optical coherence tomography[END_REF] , generally resulting in low radiance values.

If the sample moves during the acquisition of the interferometric data (especially axially, as the phase rapidly varies in that direction), the recorded interference fringes can quickly be washed out if the integration time is too high [START_REF] Sacchet | Motion artifact suppression in fullfield optical coherence tomography[END_REF] , resulting in a loss of signal. Considering in vivo imaging, it is essential that the integration time is low enough to avoid any fringe washout due to movements of the subject being imaged, especially if a system capable of in vivo imaging in common conditions with no particular means of subject stabilization (such as physical constraint or anesthesia) is desired.

Reconstruction of tomographic images

In FF-OCM, the tomographic images are the result of a combination of several phase-shifted interferometric images according to a given demodulation algorithm. Consequently, the time needed to acquire a tomographic image is at least the integration time multiplied by the number of interferometric images needed by the demodulation algorithm being used. In practice, time is also needed for the camera to transfer the data read by all the pixels during its integration time. The overall time needed to acquire the data and transfer it is referred to as the frame time of the camera, and thus the time needed to acquire a tomographic image is the frame time multiplied by the number of interferometric images needed for reconstruction. It should be noted that even though the integration time of the camera can be reduced by increasing the radiance of the light source, the actual frame time of the camera cannot always be reduced accordingly. Nevertheless, with some cameras, it is possible to define the number of pixels actually in use, and decreasing that number can reduce the frame time, as there is less information to be transferred, at the price of smaller fields being imaged or a loss in spatial resolution. Another process that can affect the speed of acquisition of tomographic images is image accumulation. Before applying the demodulation algorithm, several interferometric images corresponding to the same applied phase-shift are summed up in order to boost the interferometric signal. This increases the detection sensitivity according to Eq. ( 2), with S0 being the sensitivity when no image accumulation is performed, and N being the number of images that are accumulated:

0 10 10log ( ) S S N  (2)
At the same time, the acquisition time is multiplied by N. Image accumulation is almost always performed in FF-OCM when high quality images are desired.

Therefore, the total time to acquire a single tomographic image is the frame time multiplied by the number of images needed by the reconstruction algorithm multiplied by the number of images accumulated for each phase-shift. Note that at least three images are needed for reconstruction in order to properly demodulate the interferometric data.

Hence, even for a system for which the integration time is very low, the actual time to record a tomographic image may be significantly higher. In order to reduce it as much as possible, a two-image algorithm can be used [START_REF] Dubois | Three-dimensional cellular-level imaging using full-field optical coherence tomography[END_REF] . Although a twoimage algorithm cannot demodulate the interferometric signal (it just removes the signal background), it still gives good results regarding imaging biological samples. As mentioned previously, single shot setups, which are generally complex and costly, have also been proposed [START_REF] Moneron | Stroboscopic ultrahigh-resolution full-field optical coherence tomography[END_REF][START_REF] Dunsby | Single-shot phase stepped wide-field coherence gated imaging[END_REF][START_REF] Hrebesh | In vivo imaging of dynamic biological specimen by real-time single-shot full-field optical coherence tomography[END_REF][START_REF] Sacchet | Motion artifact suppression in fullfield optical coherence tomography[END_REF][START_REF] Schausberger | Full-field optical coherence microscopy with Riesz transform-based demodulation for dynamic imaging[END_REF] .

Regarding in vivo imaging, it should be noted that movement of the subject between successive interferometric images is less critical than during the acquisition of the interferometric data. As long as the interferometric signal has been acquired, movements between images used for reconstruction can only result in artifacts due to the recombination of signals corresponding to different zones of the subject due to its movement, that generate interferometric signals with different amplitudes [START_REF] Sacchet | Motion artifact suppression in fullfield optical coherence tomography[END_REF] . As the amplitude varies much more slowly than the phase (an error in the phase will generally not greatly affect the quality of demodulation, some algorithms insensitive to phase errors have even been developed), the constraint regarding movement over the total acquisition time is less severe than over the integration time of the camera. Nevertheless, those artifacts can significantly affect the quality of the images and should be minimized.

EXPERIMENTAL SETUP

The high speed, high resolution FF-OCM setup reported here is based on a Linnik interference microscope, the configuration that has been the most widely used in FF-OCM 5-9. It consists of a Michelson interferometer with a microscope objective in each arm, providing high transverse resolution and magnification. It has the advantage of providing flexibility regarding the choice of numerical aperture (NA) and immersion medium, while reducing light losses compared to the Mirau or Michelson interference microscope configurations, a fundamental point when high imaging speed is targeted. In order to optimize in vivo imaging, the interferometer was designed to be as compact as possible, with the aim to lay the foundations for a handheld system, as the capacity for in vivo imaging is of greater interest if it is possible to image in real-time any region of interest. Furthermore, a compact system limits motion artifacts due to external vibrations.

The designed interferometer consists in two identical microscope objectives (10x, NA = 0.3, water-immersion, Olympus) and a cube beamsplitter as small as possible, with the objectives directly connected to the beamsplitter cube housing, resulting in an interferometer with very short arms, having a total size of 11115 cm 3 and weighing 210 g, making it suitable for a future compact and light handheld setup. Silicone oil, whose refractive index typically matches the one of biological tissues, is used as immersion liquid to compensate for optical dispersion. In the object arm, the objective rests within a tank full of immersion liquid at the bottom of which a glass plate (0.5-mm-thick, made of fused silica) was placed to enable imaging the sample through it, while also permitting to press the sample against it in order to limitate sample motion as much as possible. In the reference arm, a tank full of immersion liquid is directly linked to the objective. A similar glass plate is placed at the bottom of the tank in order to compensate for the optical dispersion introduced by the plate in the object arm while also providing a 3.5% reflectivity reference surface (interface between glass and air) appropriate for FF-OCM imaging. This glass plate serving as a reference surface was placed upon a small piezoelectric (PZT) chip (5.05.02.4 mm 3 ) included within the tank, that is used for providing phase shifting in order to demodulate the interferometric signal, while the whole interferometer is placed upon a PZT stage used to set the imaging depth, and enable three-dimensional imaging. Figure 1 shows a schematic of the setup. In order to maximize the speed of the imaging system, we introduce the use of a high brightness broadband LED as the light source. Currently, phosphor LEDs having broad spectra and high radiant fluxes are starting to be commercially available. Recently, the use of a broadband LED for FF-OCM has been shown to yield identical images compared to those obtained with a conventional halogen lamp [START_REF] Ogien | High-resolution full-field optical coherence microscopy using a broadband lightemitting diode[END_REF] , nevertheless, the radiant flux of the LED was not high enough for high-speed imaging. The LED used in the present setup has a 2.3 W radiant flux, with a spectrum having a FWHM of 145 nm and a central wavelength of 600 nm, yielding an axial resolution for FF-OCM of 0.9 μm, as shown in Fig. 2. It should be noted that the spectrum is not optimal for penetration in biological tissues, as it spans over only a small part of the optical therapeutic window. Nevertheless, penetration up to a few hundreds microns is still possible. Considering the LED directivity (125° viewing angle), size (12.25 mm²) and radiant flux, it is possible to estimate its radiance to be 70 mW.m -2 .str -1 . By comparison, a typical radiance with a high-brightness halogen lamp is 10 mW.m -2 .str -1 . The LEDbased setup can achieve image rates up to 7 times superior to conventional halogen lamp based setups, if the same camera is used. Therefore this LED-based setup provides a high-speed acquisition while maintaining a high resolution, and benefits from the numerous advantages of LED illumination, including compactness, low power consumption, ease of use and maintenance, and long lifetime. As explained above, in order to benefit from the high LED radiance, the camera must also have a high frame rate (and a high FWC for high sensitivity). A CMOS camera (Photonfocus, model MV1-D1026E-160-CL) with a FWC of 200,000 e -, was used. The frame rate of the camera can be significantly increased by limiting its number of pixels in use.

Considering our LED, the integration time of the camera could be as low as 920 μs while maintaining the pixels close to saturation, resulting in a measured detection sensitivity of 68 dB. Nevertheless, the frame time was limited to ~ 7 ms if all the pixels (10241024) were in use. In order to reduce the frame time to be as close as possible to the integration time while maintaining a relevant field of view (above 200x200 μm²), 250250 pixels were used, resulting in a FOV of 250250 μm², and a frame rate of up to 1000 frames per seconds (fps). A four-frame algorithm with sinusoidal phase modulation was used for demodulation [START_REF] Dubois | Handbook of full-field optical coherence microscopy: technology and applications[END_REF] , yielding an FF-OCM imaging rate of 250 Hz. This rate enables in vivo imaging by significantly limiting motion artifacts, as explained in section 2. In order to further improve the sensitivity, it was even possible to perform image accumulation with five interferometric images while keeping in vivo imaging feasible. The resulting sensitivity was then boosted to 75 Hz, as shown in Figure 3. Experiments shown that past this number of accumulated images, no significant gain in image quality was obtained, as the gain in sensitivity was then counterbalanced by image degradation due to artifacts linked to the increased in acquisition time. With five images accumulated, the imaging rate was 50 Hz. In order to increase the signal to noise ratio of the images obtained even further, every five images were averaged together, resulting in a 10 Hz imaging rate, which can still be considered as video rate. Note that this last averaging is done after the tomographic images have been obtained, and therefore no signal can be lost during this step. Motion can just cause a blurring of the image if it is too significant during the acquisition of the five tomographic images to be averaged together.

RESULTS

In order to show the ability of this FF-OCM setup to produce images in vivo with no loss in resolution or sensitivity compared to images of immobile samples, imaging of human skin both in vivo and ex vivo was performed. In vivo skin imaging is one of the main goals of FF-OCM in order to make this imaging technique more widely used in a clinical context [START_REF] Dalimier | Full-Field Optical Coherence Tomography: A New Technology for 3D High-Resolution Skin Imaging[END_REF][START_REF] Dalimier | High resolution in-vivo imaging of skin with full field optical coherence tomography[END_REF] .

The ex vivo sample consisted of healthy human skin excised from the neck region. In vivo measurements were carried out on the author's hand. The imaging procedure consisted in pressing the skin onto the glass plate of the interferometer object arm as described above, after having placed a drop of dermatoscopy oil onto the skin in order to attenuate the reflection of the glass plate. Tomographic images were displayed in real time at a 10 Hz rate using a custom C++ software. Using the PZT stage, images could be obtained at several axial positions of interest within the epidermis. It was possible to penetrate roughly up to the superficial dermis, about 150 μm in depth. Images obtained within the stratum granulosum, stratum spinosum, and stratum basale are presented in Figure 4. A slight Gaussian blur was applied on the images for display in this paper. It should be noted that the skin imaged in vivo originated from a different location within the body than skin imaged ex vivo, resulting in some differences regarding the structure of the skin. Nevertheless, cellular level imaging is clearly demonstrated both in vivo and ex vivo, and the images are highly similar, showing that the image acquisition is rapid enough for the motion of the subject not to affect image quality. It should be emphasized that the subject being imaged was just pressing his skin onto the glass plate, with no other means of stabilization.

CONCLUSION

This work presents the implementation of a compact FF-OCM setup specifically designed to meet the needs for in vivo imaging, illuminated by a high-brightness broadband LED. This setup constitutes a simple implementation of FF-OCM, using a common demodulation process, common Linnik configuration and an inexpensive and easy to use light source, which provides high resolution in vivo imaging. It constitutes a proof of principle that in vivo imaging is possible in FF-OCM without having to resort to any complex method. Furthermore, the compactness of the introduced design paves the way for portable FF-OCM setups that will make it possible fully benefit from the capacity of FF-OCM for in vivo imaging, especially in a clinical context.

This work also constitutes a step towards the advent of LED illumination in FF-OCM, as it is shown that the high radiance of LED combined with a broad spectrum enables image rates superior to conventional thermal-light illumination, while maintaining a similar spatial resolution, and benefiting from the numerous advantages of LED illumination.

Currently, the main limitation of the system concerns the camera. Even though it is possible to reach short integration times using the LED, the frame time of the camera cannot be reduced accordingly if all of its pixels are used, impeding large field imaging. Furthermore the FWC of our camera is not high enough for high quality imaging without image accumulation, which is also a limiting factor. Those limitations could be overcome by the availability of more appropriate cameras in order to benefit from the full potential of LED illumination for high-speed FF-OCM.

Figure 1 .

 1 Figure 1. Schematic representation of the compact FF-OCM setup. L1, collector lens; L2, condenser lens; BS, beam-splitter; MO, microscope objectives; T, tanks containing silicone oil; GP, glass plates; S, sample; PZT-C, piezoelectric chip; PZT-S, piezoelectric stage; TL, tube lens; CMOS, complementary metal oxide semi-conductor camera; FG, function generator; TTL, transistor-transistor logic; PC, personal computer. The dashed rectangle represents the part of the interferometer moving vertically due to PZT S.

Figure 2 .

 2 Figure 2. a) Spectrum of the LED light source. An interference filter was used to filter out the blue component of the spectrum; b) Axial interferometric response of the system, that determines the imaging axial resolution.

Figure 3 .

 3 Figure 3. Axial response of the FF-OCM system in logarithm scale, with five images accumulated. The dashed line indicates the detection sensitivity of the system.

Figure 4 .

 4 Figure 4. Images of human skin obtained with the FF-OCM setup; a) b) and c) : in vivo, within the stratum granulosum, stratum spinosum and stratum basale, respectively; d), e) and f) : ex vivo, within the same layers, respectively. Scale bars are 50 μm.