François Lechanoine

Timothée Jacquesson

Justine Beaujoin

Barthélemy Serres

Mohammad Mohammadi

Alexia Planty-Bonjour

Frédéric Andersson

Fabrice Poupon

Cyril Poupon

Christophe Destrieux
email: christophe.destrieux@univ-tours.fr

WIKIBrainStem: An online atlas to manually segment the human brainstem at the mesoscopic scale from ultrahigh field MRI

Keywords: Brainstem, anatomy, exvivo MRI, atlas

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

Introduction

The human brainstem is one of the most densely packed areas of the encephalon, containing numerous nuclei and white matter tracts involved in awareness, motor, somesthetic, and vital functions. Its recent choice as a target for deep brain stimulation in several neurological pathologies, such as Parkinson's disease [START_REF] Okun | Deep-brain stimulation for Parkinson's disease[END_REF] or other movement disorders [START_REF] Larson | Deep brain stimulation for movement disorders[END_REF], and psychiatric diseases, such as depression or refractory obsessive compulsive disorder [START_REF] Ooms | Deep brain stimulation for obsessive-compulsive disorders: long-term analysis of quality of life[END_REF], has caused the need to comprehend its anatomy to resurface. However, microstructure and connectivity of the brainstem have mostly been studied on 2D histological stained sections [START_REF] Afshar | A variability Study[END_REF][START_REF] Nieuwenhuys | The human central nervous system[END_REF][START_REF] Nolte | The Human Brain: An Introduction to its Functional Anatomy[END_REF][START_REF] Paxinos | Atlas of the Human Brainstem[END_REF], a method that loses the 3D coherence of anatomical specimens and renders multiplanar reconstruction quite difficult. Due to these limitations and to the delicate processing of hundreds of serial histological slices, no probabilistic atlas of the whole brainstem is currently available.

Magnetic Resonance Imaging (MRI) is an appealing approach to, in a non-destructive manner, obtain 3D images of the inner structures of the brainstem. Clinical MRI scanners, using 1.5 and 3T magnetic fields, nevertheless provide images in which the internal structure of the brainstem appears relatively homogenous. Additional contrasts can be provided by more advanced sequences, for instance diffusion-weighted imaging that uses strong gradient pulses along various directions of the space that tag the spins of water molecules with phases depending on their positions, thus allowing to sensitize the NMR signal to the endogenous diffusion process.

Because it is related to movements occurring at the microscopic scale, which are strongly modulated by the presence of cell membranes acting as obstacles, the obtained diffusionsensitized signal embeds information regarding the local cellular environment that can be decoded to characterize the local tissue microstructure. Information provided by these methods helps to better delineate anatomical structures [START_REF] Nagae-Poetscher | Highresolution diffusion tensor imaging of the brain stem at 3 T[END_REF][START_REF] Salamon | Analysis of the brain-stem white-matter tracts with diffusion tensor imaging[END_REF][START_REF] Stieltjes | Diffusion tensor imaging and axonal tracking in the human brainstem[END_REF][START_REF] Virta | Visualizing and characterizing white matter fiber structure and architecture in the human pyramidal tract using diffusion tensor MRI[END_REF], but the spatial resolution of MRI clinical scanners remains relatively low -in the order of 1mm for 3D anatomical sequences and several millimeters for diffusion-weighted images. The emergence of ultra-high-field clinical MRI systems at 7T allows to extend beyond the millimeter scale in vivo both for the anatomical [START_REF] Dumoulin | Ultra-high field MRI: Advancing systems neuroscience towards mesoscopic human brain function[END_REF] and diffusion MRI [START_REF] Heidemann | Diffusion imaging in humans at 7T using readout-segmented EPI and GRAPPA[END_REF], but at the expense of longer scan durations. More recently, a novel simultaneous multi-slab, diffusion-weighted sequence was proposed to perform diffusion MRI in vivo at the submillimeter resolution at 3T [START_REF] Setsompop | High-resolution in vivo diffusion imaging of the human brain with generalized slice dithered enhanced resolution: Simultaneous multislice (gSlider-SMS)[END_REF]. However, a trade-off must be made to choose between the target diffusion sensitization magnitude and the target spatial resolution. In addition, the specific localization of the brainstem next to the petrous bones, with an important air-tissue interface causing major susceptibility artifacts, and its predisposition to movement induced by the pulsation of blood in posterior fossa arteries are all reasons that make the acquisition of very high-resolution MRI data of the brainstem extremely difficult in vivo. This limitation can partially be circumvented using preclinical magnets carrying an extreme magnetic field (11.7T), which allows a higher spatial resolution at the cost of a coil size not compatible with a whole human head. This size (about 60mm in diameter) is, nevertheless, adapted to ex vivo brainstem specimens. In addition, ex vivo specimens permit very long scanning times that are not biased by physiological noise (respiratory movements or blood pulsation) or motion [START_REF] Augustinack | Direct visualization of the perforant pathway in the human brain with ex vivo diffusion tensor imaging[END_REF][START_REF] Mcnab | High resolution diffusion-weighted imaging in fixed human brain using diffusion-weighted steady state free precession[END_REF][START_REF] Miller | Diffusion imaging of whole, post-mortem human brains on a clinical MRI scanner[END_REF]. Conversely, death and specimen fixation induce dramatic changes in T2 relaxation time and diffusion coefficients (D'Arceuil and de Crespigny, 2007;D'Arceuil et al., 2007;[START_REF] Leprince | Optimization of sample preparation for MRI of formaldehyde-fixed brains[END_REF], such as post-mortem tissues depicting diffusion coefficients two to five times lower than in vivo conditions (D'Arceuil et al., 2007). This must be instrumentally compensated by more powerful gradients -also available in preclinical scanners -to obtain access to high diffusion sensitizations (up to 10,000s/mm²) while keeping the echo time very short (typically smaller than 20ms).

Only a few papers have been published on ultra-high field MRI brainstem anatomy (Calabrese et al., 2015a;[START_REF] Fritz | Ultra-high resolution and multi-shell diffusion MRI of intact ex vivo human brains using kT-dSTEAM at 9.4T[END_REF][START_REF] Rushmore | 3D Exploration of the Brainstem in 50-Micron Resolution MRI[END_REF], usually on single subjects, with a limited number of more or less precisely described anatomical classes. This renders difficult the replication of the results on other specimens. One of the explanations, apart from the high cost of such scans, is the lack of a precise anatomical reference usable for ex vivo MRI.

The goal of this paper is to propose a set of rules to manually segment ultra-high/extreme field (11.7T) ex vivo MR images of the human brainstem in approximately 120 hierarchically organized anatomical classes. These rules were assembled in a publicly available web-based application, WIKIBrainStem (https://fibratlas.univ-tours.fr/brainstems/index.html), providing a 3D representation and a set of rules for reproducibly segmenting the brainstem. Such a tool is a mandatory step towards an automated atlas of the human brainstem.

Materials and Methods

Specimen preparation and acquisition

One brainstem was obtained from body donation from a subject without a history of neurological or vascular diseases. The brain was extracted less than 24 hours after death and fixed in a 10% formalin solution in distilled water for 3 months. The brainstem was then split from the cerebellum and diencephalon to obtain a specimen compatible with the coil size (60mm in diameter). To partially compensate for effects of formalin fixation by increasing the T2, T2* and diffusion coefficient, the specimen was soaked for 3 weeks in a 0.1M phosphate-buffered saline solution (PBS) at 4°C prior to scanning [START_REF] Leprince | Optimization of sample preparation for MRI of formaldehyde-fixed brains[END_REF]. A specific cylindrical container was manufactured to exactly fit the inner diameter (60 mm) of the MR coil and to avoid air bubbles responsible for imaging artifacts. To stabilize its temperature at 20°C, the specimen was positioned within this container and transferred to the magnet room 4 to 5 hours prior to scanning. This was required to limit effects related to temperature variations that possibly induce modifications in the local T2 and the local apparent diffusion coefficient (ADC) [START_REF] Thelwall | Effects of temperature and aldehyde fixation on tissue water diffusion properties, studied in an erythrocyte ghost tissue model[END_REF]. To avoid specimen desiccation during acquisition and to reduce imaging artifacts, the sample was immersed in a proton-free fluid (Fluorinert, FC-40, 3M Company, USA). This fluid does not provide any signal during image acquisitions and has a susceptibility coefficient similar to that of brain tissue, thus reducing susceptibility-related imaging artifacts.

The specimen was then scanned using a preclinical 11.7T Bruker MRI system (BioSpec 117/16 USR Bruker MRI, Etlingen, Germany) equipped with a strong gradient system (maximum gradient magnitude = 780mT/m, slew-rate = 9660T/m/s), as well as a 60mm transmit/receive volume coil. The choice of a volume-versus-surface coil was motivated by the need for a homogeneous excitation profile over the large field of view of the brainstem despite a reduced signal-to-noise ratio with volume coils. The B1 field homogeneity also comes with a constant noise model over the field of view, which is also recommended for the noise removal of diffusion-weighted MRI data. The imaging protocol included anatomical and diffusion scans.

Anatomical images were acquired using a 3D T2-weighted spin-echo sequence with isotropic spatial resolution of 100 µm and 185 µm, matrix 500x512; 800 slices, TR/TE=500/20 ms, and a total scan time of 35h33min. A single-shell High Angular Resolution Diffusion Imaging (HARDI) dataset was acquired using a conventional 2D Pulsed-Gradient Spin-Echo (PGSE) sequence. The b-value, characterizing the gradients used to measure diffusion, was chosen to 4500 s/mm 2 to compensate for post-mortem loss of contrast. Diffusion data were acquired along 400 directions uniformly distributed over a sphere and split into 12 blocks of 31 directions and one of 28 due to memory limitation of the MRI system and to limit heating of the specimen, which is prone to induce artifacts. For each block, two b=0 images (without diffusion ponderation) were acquired.

Scanning parameters were as follows: isotropic spatial resolution of 300µm; TR/TE= 9000/24.6ms; Δ/δ= 14.4/4.3ms; matrix size: 167x163; 250 slices; and a total scan time of 5 days and 18 hours. The diffusion tensor was calculated for each voxel using a weighted least-squares approach to generate various tensor derived scalar maps including fractional anisotropy (FA), mean diffusivity (MD), and color-encoded direction (CED -RGB code) maps. Microstructural and connectivity data obtained from the HARDI acquisition were not used in the frame of the current study focused on anatomical structures, but they will be investigated in another study.

Preprocessing of anatomical and diffusion MRI dataset was done using the freely available Ginkgo ToolBox (Poupon, n.d.) providing a set of processing tools to correct for imaging artifacts and a set of tools to specifically process diffusion MRI data. Anatomical and diffusion MRI datasets were processed at their original resolutions (100µm and 185µm for the anatomy and 300µm for diffusion respectively). First, the 185µm resolution anatomical MRI dataset and the 300µm diffusion MRI dataset at b=0s/mm² were matched to the highest resolution anatomical dataset (at 100µm), using the 3D affine registration Ginkgo tool relying on the optimization of a mutual information criterion optimized using coarse-to-fine strategy and involving a Nelder-Mead simplex algorithm [START_REF] Wells | Multi-modal volume registration by maximization of mutual information[END_REF]. No intensity correction was applied to the anatomical scan to avoid any modification of the raw dataset intensity to be manually segmented. The 12 Diffusion MRI datasets were corrected for noise using the Non Local Means filter of the Ginkgo toolbox assuming a Rician noise model (related to the single-channel transmit-receive coil used to perform the acquisitions) [START_REF] Wiest-Daesslé | Rician noise removal by non-Local Means filtering for low signal-to-noise ratio MRI: applications to DT-MRI[END_REF]. The diffusion MRI acquisitions were performed using a multi-shot PGSE sequence and a high number of shots (33 shots), which allowed to reduce the geometrical distortions related to susceptibility effects or eddy currents to a level which does not require the application of any dedicated correction and, thus, preventing the use of any resampling of the data. Lastly, rotationally invariant DTI maps including the apparent diffusion coefficient (ADC), the fractional anisotropy (FA), the transverse (Dparallel) and longitudinal (Dperpendicular) diffusion coefficients were computed with a further color-encoded direction map (CED) using a robust fit to ensure the positiveness of the solution [START_REF] Fillard | Clinical DT-MRI estimation, smoothing, and fiber tracking with log-Euclidean metrics[END_REF]. The superimposition of the CED map to the high-resolution anatomical scans contributed to enhance the delineation of brainstem structures while keeping each modality to its native imaging resolution.

Definition of anatomical classes

We created a highly detailed hierarchical parcellation scheme of the brainstem from a literature review including macroscopic and histologic textbooks [START_REF] Brodal | The central nervous system: structure and function[END_REF][START_REF] Kamina | Anatomie Clinique[END_REF]Naidich and Duvernoy, 2009;[START_REF] Nieuwenhuys | The human central nervous system[END_REF][START_REF] Paxinos | Atlas of the Human Brainstem[END_REF]. Brainstem nuclei and sub-nuclei found in the literature were included in a database without knowing a priori if they would be visible on the MR data. Some structures not belonging to the brainstem but partially included at the border of the segmented specimen (cerebellar and diencephalic nuclei) were added to this database. A five-digit hierarchical index was assigned to each anatomical class and was stored in the voxels of the segmented volume (Table 1):

-The first digit designated the right (1), left (2) or median location of the structure/substructure.

-the second digit defined the main group to which the structure belonged: (1) proper nuclei of the brainstem, (2) cranial nerve nuclei, (3-5) reticular nuclei, (6) cerebellar and diencephalic nuclei; -the third digit was related to the location of the structure within these groups; for instance, the proper nuclei of the brainstem were subdivided into mesencephalic (third digit = 0-2), pontine (third digit = 3-5) and medullary nuclei (third digit = 6-9); -the fourth digit designed an anatomical structure; -finally, a fifth digit equal to "0" was used to consider this structure as a whole; if this structure was subdivided into different anatomical classes, non-null values of the 5th digit where used; for instance the substantia nigra (1/21060) was subdivided into compact (1/21061) and reticular (1/21062) parts;

For example, the right compact part of the substantia nigra was labeled 11061:

Manual segmentation -Definition of segmentation rules

The manual segmentation of the obtained 800 slices was performed using an iMac computer (3,5 GHz Intel Core i7) running High Sierra connected to a combined 27-inch LCD monitor and graphic tablet and stylus (Wacom Cintiq 22HD). We used the Freeview software, part of the Freesurfer package (https://surfer.nmr.mgh.harvard.edu/), which simultaneously displayed coronal, axial, and sagittal planes, plus a 3D rendering of the segmented classes; co-registered anatomical (T2 100µm and T2 185µm) and diffusion (ADC, FA and CED) maps were superimposed with a user-defined transparency (offset) to obtain an optimal delineation for each anatomical class.

Anatomical classes were sequentially segmented, beginning with the biggest and easily delineable ones (for instance, principal nucleus of the inferior olive or red nucleus) before moving to smaller or ill-limited nuclei (reticular nuclei). A dual-pass process included a first rough segmentation on images with a sufficient diffusion-weighted/T2-weighted contrast (diffusion and/or 185µm T2), followed by a refinement step using higher-resolution T2weighted contrast alone (100µm). Each anatomical class was attributed a specific RGB color stored in a dedicated look up table (LUT). Part of the structures listed in the original LUT were The segmentation and rules definition were initially performed by one author (FL) and reviewed and adapted by two additional ones (TJ, CD). Each ROI of the segmentation was finally regularized using a 3D approach to discard irregularities stemming from the manual segmentation task: a binary volume was created for the background for each anatomical class; then, these binary volumes were smoothed down (mean filtering with gaussian kernel, sigma= 0.5 mm), each voxel containing the probability to belong to the corresponding anatomical class.

For each voxel, the anatomical class having the highest probability was searched for acrossindividual files (anatomical classes and background). The resulting smoothed map was used to build the wiki.

WIKIBrainStem interface

WIKIBrainStem (https://fibratlas.univ-tours.fr/brainstems/index.html) is a publicly available WebApp specifically developed by ILIAD3 for this project. It is composed of two components: a 3D viewer and a media wiki. The first one (Figure 1) is based on an open source, webGL-based javascript library (https://github.com/xtk/X). This graphical user interface allows for real-time 3D navigation through the segmented results, represented as vtk files (https://vtk.org/). The proposed anatomical hierarchy is displayed using a tree-like menu on the right panel of the application. While the user browses this hierarchical menu, the alpha translucency of the selected structures is modulated to highlight them in a 3D view on the middle panel of the application. This 3D view can be zoomed in and out, and the position of the camera can be interactively changed. Finally, the left panel lists the structures highlighted in the 3D view and contains links pointing to the corresponding wikipages, which are displayed as a pop-up when selected. -basics of its anatomy, function and main connectivity;

-a link to the widely used Uberon ontology [START_REF] Mungall | Uberon, an integrative multi-species anatomy ontology[END_REF] entry for this structure.

-the optimal combination of MRI sequences for segmenting this anatomical class (used MR sequence(s) and their related transparency/offset);

-the rules to roughly localize this structure, relatively to surface landmarks or easily recognizable internal structures;

-a precise description of the limits of this structure, related to changes in the various MR contrasts, to surrounding structures or reference planes when no other clear limit was visible;

-and the difficulties we faced during this class segmentation (lack of clear descriptions of its boundaries in the literature, difficulties in visualizing its limits on MR images, choices we were required to make in case of uncertainty…);

Data availability

The NIFTI T2-weighted 100 and 185µm isotropic anatomic images, the scalar maps derived from diffusion-weighted imaging (FA, CED), and the segmented volume will be freely available under

Creative Commons CC BY-NC-SA 4.0 license on the Fibratlas project website (https://fibratlas.univ-tours.fr/), along with the LUT table, as well as on the Human Brain Project atlas webpage (https://ebrains.eu/services/atlases/brain-atlases/). This format allows the use of the data by any modern imaging software, voxels of the segmented volumes containing the index value of the corresponding anatomical class.

Ethics -Conflicts of interest

The anatomical specimen used in this study was obtained from the Tours body donation program. The donor had previously consented to the terms in which his body would be used for academic or research purposes. The authors do not have any conflict of interest with other parties or organizations that could inappropriately influence their work.

Results

11.7T MRI acquisition and data processing

Anatomical 100µm T2 3D Multi Slice Multi Echo series (MSME3D) sequence (Figure 3a andd) provided highly detailed images on which very small nuclei were possibly detected, for instance the trochlear nucleus or the accessory nuclei of the inferior olive. However, the cost of such a high resolution was a loss of contrast, which sometimes decreased our ability to distinguish two neighboring nuclei. For this reason, we sometimes were required to use anatomical images with a lower resolution (185µm) but a higher signal/contrast-to-noise ratio. Major tracts were easily visible on T2 anatomical sequences thanks to their lower signal related to their important myelin content associated with a shorter T2 relaxation time, whereas nuclei appeared in hypersignal, with various shades of gray. Diffusion sequences: ADC, FA and color-encoded direction maps were available at a 300µm isotropic resolution. A favorable contrast between tracts and nuclei was usually present on FA maps, on which the nuclei were black (corresponding to low FA values, possibly due to lower number of fibers or fibers having multiple directions), while the white matter tracts were white (corresponding to high FA values). Nevertheless, the white matter was quite homogeneous on FA maps, making it difficult to precisely delineate the main white matter pathways. FA map was especially useful to segment reticular nuclei, since it showed the intermediate reticular zone

(1/23300), which is the main organizer for the classification of the reticular formation proposed by Paxinos [START_REF] Paxinos | Atlas of the Human Brainstem[END_REF].

The numerical definition of the optimal contrast windowing for each modality was impossible, because it usually varies at both extremities of the specimen due to a signal drop and may vary across specimens and imaging sessions and parameters. Besides these instrumental limitations, the optimal contrast also depends on the anatomical environment of the segmented anatomical class. For instance, for a given nucleus it should be adapted across slices depending on the density of the surrounding white matter tracts. We, therefore, decided not to provide any numerical value, but to display a large number of figures on WikiBrainStem website to illustrate what we consider a contrast suitable for segmentation.

The overlay rendering of T2-weighted and scalar maps obtained from diffusion was of great assistance in delineating some of the anatomical classes (Figure 3c and f), especially information on orientation and localization of the main white matter pathways derived from CED maps. For example, given their cranio-caudal predominant orientation, the corticospinal tract, medial lemniscus and medial longitudinal fasciculus were displayed blue on CED maps. These fasciculi were not labeled in the atlas but were very valuable landmarks for nuclei segmentation.

Segmentation

Definition of anatomical classes from the literature, description and improvement of the proposed anatomical rules, redaction of the WIKIBrainStem pages and, finally, segmentation required around 900 hours for the first anatomical expert. Revision of the rules and pages by the two other experts needed approximately 500 additional hours.

Table 1: number of anatomical structures

The table lists the number of anatomical structures and substructures retained from the literature and currently segmented (in parentheses). Since the subdivided structures may be generated from their divisions, only the substructures and non-subdivided structures (in red) were included in the LUT, for a total 115 anatomical classes: 44 for the proper nuclei of the brainstem (A), 33 for the cranial nerve nuclei (B), and 38 for the reticular formation and related structures (C).

99 brainstem structures defined from the literature were initially retained, from which 29 were subdivided in 80 substructures (Table 1-2). Out of these, it was possible to segment 99 structures and 29 substructures from the MR dataset, and a total of 115 brainstem anatomical classes were included in the LUT (see Table 1 for details). Additionally, 8 structures of the diencephalo-mesencephalic junction and 8 structures of the cerebellum, located at the border of the anatomical specimen, were partially segmented and included in the LUT but not described in the WIKIBrainStem pages. The hierarchical tree of these structures is presented in Table 2 and the volume of each fully segmented anatomical class is given in Table 3 (supplementary material). All the anatomical classes (structures and substructures) initially retained from the literature are displayed. The ones currently segmented from the dataset are displayed with the color used to display them within the atlas. Structures displayed with a dotted pattern were not directly segmented, but can easily be reconstructed from their segmented substructures. The name of each segmented class is followed by the corresponding abbreviation used in the atlas as defined by Paxinos [START_REF] Paxinos | Atlas of the Human Brainstem[END_REF], and by a letter showing if median (M), right (R) or left (L) structure is available. Groups of anatomical classes are followed by the range of indices of the anatomical classes they contain. The first digit of the index represents the right (1), left (2) or median (3) location of the structure ("x" as first digit of the index is generic for laterality)

Anatomical rules

Anatomical rules are detailed in the wiki for each of the segmented structures/substructures. As an illustration, we present here the segmentation protocol for 4 nuclei to segment with various degrees of difficulty: two easy (red nucleus and principal nucleus of the inferior olive), one difficult (facial n.), and one medium (Locus Coeruleus).

Example 1 (easy to segment nucleus): red nucleus

The red nucleus (R), located within the tegmentum of the mesencephalon, was easily segmented because of its elliptical shape and the presence of dense, surrounding tracts providing a strong contrast. Despite initially retained from the literature review, its parvocellular and magnocellular parts were not visible enough to be specifically segmented. It was labeled 1/21100: It was best observed from FA/CED maps overlaid (50%) onto T2-weighted 100µm map (100%). It was first roughly localized within the tegmentum of the mesencephalon, on an axial view running through the rostral third of the superior colliculus. It appeared as an ovoid structure having a diameter of about 7mm. Its limits were defined in every direction as shown in Figure 4.

The limits of round or oval structures, such as R, were then roughly drawn on non-contiguous axial slices to obtain a "pattern", similar to a loose cage or wicker basket (Figure 5). The latter helps to achieve the segmentation on other planes. A: non-contiguous slices were segmented mainly form the axial slices. B: resulting "patterns" of both red nuclei, similar to cages or wicker baskets at 2 different steps of segmentation. C: when viewed from another section plane, here coronal, these "patterns" appear as discontinuous dots which help the segmentation of lower contrast areas (for instance caudal pole on this view).

Example 2 (easy to segment nucleus): principal nucleus of the inferior olive

The principal nucleus of the inferior olive (IOPr), located within the ventral medulla oblongata, was also easy to segment due to clear-cut limits on anatomical 100µm images, given many surroundings fascicles. It was labeled 1/21651:

The best combination of MRI used to segment the IOPr was an overlay of CED map (50% transparency) onto T2-weighted MRI at 100µm (100% opacity). It was easy to localize within the ventral part of the rostral medulla oblongata, underneath the surface of the olive, just lateral to the pyramids, thanks to its typical shape evoking a dorsomedially opened purse. The limits of IOPr are detailed in Figure 6, and a 3D rendering provided Figure 7.

Example 3 (nucleus of intermediate segmentation difficulty): locus coeruleus

The locus coeruleus (LC) is a major noradrenergic reticular nucleus of the rostral and mid pons,

Example 4 (difficult to segment nucleus): Facial nucleus (7N)

7N was more difficult to segment because it was not outlined by surrounding white matter tracts and was in close proximity with the reticular formation of the pons, without any clear contrast.

Moreover, facial nerve fibers left the dorsal aspect of the nucleus to curve around the dorsal and then medial aspects of the abducent nucleus, forming the facial colliculus. This isodirectional population of fibers locally increased the FA at the dorsal aspect of the nucleus, making its precise segmentation even more difficult. 7N was identified as 1/22110:

The 7N was first localized using CED or T2 weighted 185µm maps (Figure 12). It was then finely segmented using anatomic T2-weighted MRI at 100µm with strong contrast overlaid by CED map (50% transparency). CED/T2 185µm maps were used for rough location of 7N: an axial slice running just rostral to the pontomedullary sulcus, through the center of the abducens nucleus (6N) was selected. The facial nerve (7n) appeared as a black linear structure with a ventromedial curve; 7N was round and medial to the 7n. It appeared violet-blue on CED map and was delineated from the 6N by a blue darker area (intermediate reticular zone, IRt). Then, the limits of 7N were defined:

• cranial and caudal: IRt;

Discussion

This paper is the first description of a systematic protocol for brainstem segmentation at the mesoscopic scale from multi-modal MRI contrasts acquired at 11.7T: T2-weighted images at various resolutions, as well as quantitative maps stemming from diffusion-weighted imaging.

Ex vivo MRI is a unique way to non-destructively explore anatomical specimens without losing their 3D coherence. Its use is, nevertheless, questionable since it has its own limitations in terms of spatial resolution and contrast and can be biased by an unknown amount of tissue deformation/artifacts. The choice of optimal imaging data used for segmentation was, indeed, a compromise between spatial resolution and contrast, which is characterized by the signal-tonoise ratio (SNR) and contrast-to-noise ratio (CNR). Among other parameters, the SNR depends on the acquisition time, field strength, and volume of the voxel. For example, doubling the voxel size, from 100µm to 200µm isotropic resolution, would multiply the SNR by a factor of 8. This would provide 8 times more signal and, consequently, more contrast to noise ratio. Ex vivo imaging benefits from an unlimited scanning time and absence of physiological noise but suffers from a severe drop in T1, T2 and diffusion coefficients that occurs after death and during the fixation process in formalin (D'Arceuil and de Crespigny, 2007;D'Arceuil et al., 2007;[START_REF] Leprince | Optimization of sample preparation for MRI of formaldehyde-fixed brains[END_REF]. Immersion in PBS for one week prior to scanning partially reverts this trend [START_REF] Leprince | Optimization of sample preparation for MRI of formaldehyde-fixed brains[END_REF] but is not efficient enough to obtain usable mesoscopic resolution images in conventional scanners. Using preclinical MRI systems that provide higher values of magnetic field -but also much higher gradient strengths -is, therefore, an appealing solution to study the brainstem anatomy. For instance, the feasibility of a 3D DTI acquisition of the brainstem at 11.7

T has been shown with 125-255 µm isotropic resolution [START_REF] Aggarwal | Feasibility of creating a high-resolution 3D diffusion tensor imaging based atlas of the human brainstem: A case study at 11.7[END_REF]. This seminal paper was, nevertheless, limited by coil size considerations, because only the medullary-spinal junction was scanned at the highest resolution (125µm isotropic), whereas the resolution for the whole specimen was 255µm isotropic. Moreover, the same study used a relatively limited b value and number of directions (4000s/mm 2 , 30 directions at 255 µm; 2100 s/mm 2 , 16 directions at 125 µm), as compared to the one we used (4500/7500/10000 s/mm 2 , 400 directions), which allows more elaborated modeling of the diffusion process than the simple 6-element tensor model used in [START_REF] Aggarwal | Feasibility of creating a high-resolution 3D diffusion tensor imaging based atlas of the human brainstem: A case study at 11.7[END_REF]. Despite these limitations, this study was able to show a proper correspondence to myelinstained sections. This was later confirmed at 7T (Calabrese et al., 2015b), with a 50 µm (3D gradient echo pulse sequence) and 200µm (diffusion data, 4000 s/mm 2 , 120 directions) isotropic spatial resolution.

To increase the signal-to-noise ratio while preserving the in-plane spatial resolution, most of the brainstem postmortem anatomical and diffusion studies use anisotropic voxels by increasing the slice thickness (Naidich and Duvernoy, 2009;[START_REF] Prats-Galino | Functional anatomy of subcortical circuits issuing from or integrating at the human brainstem[END_REF][START_REF] Soria | Improved assessment of ex vivo brainstem neuroanatomy with high-resolution MRI and DTI at 7 Tesla[END_REF]. This provides very precise images in the acquisition plane but does not allow for multiplanar nor 3dimensional reconstructions and, consequently, limits the fine segmentation of internal structures. We deliberately used another approach based on multi-resolution, multi-weighted images; acquisitions were performed at different spatial resolutions and, for some of the segmentation, we used less resolved sections with an enhanced contrast, different weightings (T2, diffusion-derived quantitative maps) or a combination of different images. This method resulted in a combination of adequate spatial resolution and contrast, while preserving 3D reconstructions of the images.

Ex vivo MRI-artifacts were used to recognize some anatomical classes; for instance, the melaninrelated hypo-signal was a suitable marker for the substantia nigra and nucleus coeruleus. Most artifacts were, nevertheless, undesired, such as small air bubbles responsible for susceptibility artifacts at the surface of the specimen. Other artifacts observed at the fomblin © -brainstem interface were especially important at higher resolutions (100µm) in areas of small protuberances, such as the caudal part of the inferior colliculus or the upper part of the olive.

They led to a certain degree of misregistration of diffusion onto anatomical series at the periphery of the specimen, compelling us to use less resolved T2-weighted images in these areas (185 µm). However, diffeomorphic registration could be used to improve the alignment of diffusion and anatomical scans in the future.

The proposed anatomical classes have been, of course, previously described in the anatomical or radiological literature [START_REF] Brodal | The central nervous system: structure and function[END_REF][START_REF] Kamina | Anatomie Clinique[END_REF]Naidich and Duvernoy, 2009;[START_REF] Nieuwenhuys | The human central nervous system[END_REF][START_REF] Paxinos | Atlas of the Human Brainstem[END_REF]. Nonetheless, to our knowledge, this is the first time that an exhaustive and hierarchical list of anatomical classes has been proposed, associated with step-by-step detailed segmentation protocols.

Every anatomical hierarchical classification is a matter of convention. For instance, Watson [START_REF] Watson | Time for Radical Changes in Brain Stem Nomenclature-Applying the Lessons From Developmental Gene Patterns[END_REF] recently proposed to replace the classical morphological mesencephalon, metencephalon, and myelencephalon classification with a more current form based on genetics, with a more limited extension of the pons. In this work, we chose a classical subdivision of the gray matter of the brainstem, which has anatomical and practical justifications; classes were derived from the Terminologia Anatomica (Federative Committee on Anatomical Terminology, 1998) and major textbooks (Naidich and Duvernoy, 2009;[START_REF] Nieuwenhuys | The human central nervous system[END_REF][START_REF] Paxinos | Atlas of the Human Brainstem[END_REF], and were logically classified regarding their anatomical location and function. This method also allowed an easy concatenation of nuclei from a given region; Table 2 illustrates the hierarchical tree of the segmented structures and provides the range of index at each level of the classification; for instance, proper nuclei of the mesencephalon located on the right side (first digit 1), left (first digit 2) side, or on the midline (first digit 3), can be pooled by simply maintaining index classes starting at (1/2/3)10, (1/2/3)11 or (1/2/3)12. Additionally to this hierarchical classification, we also described the optimal MRI sequences and the anatomical rules used for this segmentation and rendered this information publicly available through a web application and site. The goal here was to provide a simple protocol, usable on other subjects and by other teams. The original list contained 123 structures, from which 29 were subdivided into 80 substructures. We purposely attempted to be as exhaustive as possible in the list of anatomical classes, to be able to extend our protocol using alternate imaging methods, such as polarized light imaging or optical coherence tomography in the future. Consequently and due to the limited resolution of MRI, only 99 structures were segmented, from which 13 were subdivided in 29 substructures. Despite these limitations, our parcellation is the most complex proposed so far for ex vivo MRI of the brainstem. For example, in a recent paper Rushmore [START_REF] Rushmore | 3D Exploration of the Brainstem in 50-Micron Resolution MRI[END_REF] proposed a parcellation of the brainstem specimen previously scanned by Calabrese using a 7T preclinical scanner (Calabrese et al., 2015b). Images were acquired at a spatial resolution of 50µm isotropic, but the segmentation was performed after down sampling to 250µm. This resulted in the segmentation of the brainstem in only 25 gray matter anatomical classes (50 for both sides). Some anatomical classes initially retained in the list of potentially segmentable structures were finally not segmented because they were too small to be seen on MRI (often accessory nuclei) due to a lack of contrast with the surrounding structures, or because they were only recognizable at a cellular, neurotransmitter or electrophysiological level. The classification of the reticular nuclei was especially difficult due to their complex and ill-defined anatomy. We, nevertheless, used the classification employed by Paxinos [START_REF] Paxinos | Atlas of the Human Brainstem[END_REF], since the intermediate reticular formation was visible from FA maps. Similarly, some structures located at the field of view's border were only partially segmented: dentate nucleus, medial geniculate body, peripeduncular nucleus, subthalamic nucleus, spinal part of the trigeminal nucleus addition of extra data than a direct validation; since these methods use different physical phenomena (optical transmission/attenuation, birefringence, magnetic resonance), a perfect correspondence between the borders of a given anatomical structure studied by different methods seems unlikely at a high resolution, despite a rough match being found at a lower resolution [START_REF] Solsberg | MR Imaging of the Excised Human Brainstem: A Correlative Neuroanatomic Study 11[END_REF]. Moreover, a comparison to histology would not completely solve the problem, because ambiguities remain even for the segmentation of immuno-histochemical stained slices [START_REF] Brodal | The central nervous system: structure and function[END_REF][START_REF] Kamina | Anatomie Clinique[END_REF]Naidich and Duvernoy, 2009;[START_REF] Nieuwenhuys | The human central nervous system[END_REF][START_REF] Paxinos | Atlas of the Human Brainstem[END_REF]. The structure of WIKIBrainStem will allow its future extension to non-MRI data that may arise from additional studies to be very feasible. In the same vein, the segmentation rules described in this study were developed for the field strength and imaging protocols detailed in the Materials and Methods section. Different acquisition methods, more or less sensitive to the underlying microstructure, as well as the impact of different techniques for specimen preparation, may thus lead to different results. For instance, the volume of the red nucleus, still considered as easy to segment, was measured by several authors [START_REF] Eapen | Using High-Resolution MR Imaging at 7T to Evaluate the Anatomy of the Midbrain Dopaminergic System[END_REF][START_REF] Keuken | Effects of aging on T1* , T2* , and QSM MRI values in the subcortex[END_REF][START_REF] Keuken | Quantifying inter-individual anatomical variability in the subcortex using 7 T structural MRI[END_REF][START_REF] Zou | Regional morphometric abnormalities and clinical relevance in Wilson's disease: regional morphometric abnormalities in WD[END_REF] and ranged from 107 to 321 mm 3 , depending on the used magnetic fields and imaging protocols (right and left red nuclei were measured to 142 and 139 mm3 respectively in our study). Assessing the influence of the imaging parameters, field strength, and specimen preparation methods is far beyond the scope of this work, but the proposed anatomical rules may need to be adapted to very different imaging conditions.

The brainstem is usually considered less variable than the more rostral parts of the encephalon, such as the telencephalon. However, no precise data is currently available on this topic. Although the present anatomical rules were defined from a single anatomical specimen, the definition of each anatomical class greatly depends on its anatomical environment, such as the surrounding tracts and nuclei. Consequently, even if the same region is slightly variable in other specimens, the anatomical rules will be usable. One of the next applications of WIKIBrainStem will be the segmentation of several anatomical specimens by different experts. This will produce probability maps of the brainstem inner structures across subjects, which will be the starting point to propose an automated segmentation protocol, as this has already been performed at lower resolution for the brain [START_REF] Destrieux | Automatic parcellation of human cortical gyri and sulci using standard anatomical nomenclature[END_REF]. Such a probabilistic atlas of the brainstem defined at very high resolution will later be used for an automated parcellation of lower resolution images obtained from clinical MRI systems (1,5 or 3T). This would lead to a probabilistic localization of the nuclei from in vivo data at a resolution at which they are obviously not visible. The registration process mapping the atlas to individual in vivo datasets will clearly need to take into account possible distortion related to the specimen extraction and formalin-fixation. For instance, formalin fixation of the brainstem induces a shrinkage of 8.3% in the longitudinal direction, whereas no change is observed transversally [START_REF] Quester | The shrinkage of the human brain stem during formalin fixation and embedding in paraffin[END_REF].

Conclusion

The protocol of segmentation of the whole brainstem that we developed from high resolution, ex vivo, ultra-high field MRI at 11.7 Tesla is the most detailed to date. The proposed segmentation rules were gathered in a web-based application and site, which are freely available to the public. This represents a first step towards the creation of a multi-subject probabilistic atlas of the brainstem, applicable to automatically segment high-resolution, but also clinical images. This would certainly help with the precise localization of brainstem lesions (vascular, traumatic or tumoral), choice of the most appropriate surgical approaches and to precisely delineate anatomical targets for deep brain stimulation.

des dons du corps du Centre Ouest, Tours, who made this study possible by generously donating his body to scientific research. We also acknowledge Mr Gérald Deluermoz (université de Tours) who extracted the anatomical specimen and Rudolf Hergesheimer for editing the manuscript.

Funding: this project/research has received funding from the European Union's Horizon 2020 Framework Program for Research and Innovation under the Specific Grant Agreement No. 945539 (Human Brain Project SGA3). It also benefited from a specimen collected in the frame of the 2014 Agence Nationale de la Recherche 14-CE17-0015-01 project. FL was funded for one year by the Agence Régionale de Santé Centre-Val-de-Loire.

  they were not yet clearly distinguishable on the datasets. The part of cerebellar and diencephalic nuclei located within the anatomical specimen was also segmented.

  Figure 1: Main page of WIKIBrainStem A: main window showing an interactive 3D model of the selected structures and brainstem surface. B: hierarchical tree of the main structure groups; C: list of the displayed structures pointing to the corresponding wiki pages.Original application available at : https://fibratlas.univtours.fr/brainstems/index.html

-Figure 2 :

 2 Figure 2: Example of a wiki page for the red nucleus. Each page gives the following information: A, anatomical details; B, used datasets; C, segmentation rules; D, difficulties; E, 3D model. Original page available at: https://fibratlas.univtours.fr/mediawiki/index.php/Red_nucleus

Figure 3 .

 3 Figure 3. Acquisitions of the brainstem with the Bruker 11.7T preclinical MRI system. a-c) axial slices; d-f) sagittal slices a, d) anatomical T2 weighted series; 100µm isotropic voxels. Major tracts were easily visible as low signal areas, whereas nuclei appeared lighter, with various shades of gray. b, e) Diffusion Tensor Imaging (DTI) color-encoded maps; 300µm isotropic voxels. Indicators at the bottom right show the color encoding for fibers main direction. d, f) 50% overlay of color encoded maps onto anatomical T2 series.

Figure 4 :

 4 Figure 4: segmentation rules for the red nucleus (R). R was segmented thanks to the following limits: • cranial: thalamus (Th, better seen in green on the sagittal plane) and third ventricle (3V) rostro-medially. • caudal: enclosed within the superior cerebellar peduncle (scp) containing the dentato-rubro thalamic tract, which is "Y" shaped and blue on sagittal view (*). On axial view, the nucleus is circled by this tract (blue), • ventro-lateral: substantia nigra (SN, black, on axial), • ventro-medial: interpeduncular fossa (IPF), • dorsal: superior cerebellar peduncle (scp) and PAG (grey), • medial: ipsilateral intra axial course of the oculomotor nerve (3n, green, axial). A, B, C: respectively axial, sagittal and coronal slices, overlay of T2 weighted 100 µm (100% opacity) and CED map (50% transparency). D, E, F: same segmented slices.

Figure 5 :

 5 Figure 5: construction of the red nucleus "pattern".A: non-contiguous slices were segmented mainly form the axial slices. B: resulting "patterns" of both red nuclei, similar to cages or wicker baskets at 2 different steps of segmentation. C: when viewed from another section plane, here coronal, these "patterns" appear as discontinuous dots which help the segmentation of lower contrast areas (for instance caudal pole on this view).

Figure 6 :

 6 Figure 6: segmentation rules for the principal nucleus of the inferior olive (IOPr)IOPr was first localized just lateral to the pyramids (Pyr), underneath the olive (Oli). It was easily segmented thanks to its characteristic shape and to the contrast to the surrounding structures:• externally: the amiculum (ami), forming an external capsule, • internal: the hilum (hil), easily limited since it contains the spino-olivary tract which appears reddish. A, B, C: respectively axial, coronal and sagittal slices, overlay of T2 weighted 100 µm (100% opacity) and CED map (50% transparency). D, E, F: same slices on T2 weighted 100 µm. G, H, I: same segmented slices.

Figure 7 :

 7 Figure 7: Principal nucleus of the inferior olive (IOPr) overlaid onto sagittal plane.

  which was labeled 1/23850 It was best seen on T2-wieghted MRI dataset at 100µm, on which it appears as a juxtaposition of white and black dots, resulting from an artifact induced by its neuromelanin content[START_REF] García-Lorenzo | The coeruleus/subcoeruleus complex in rapid eye movement sleep behaviour disorders in Parkinson's disease[END_REF]. It was almost not visible at a lower resolution. The proposed segmentation rules include a localization step (Figure8) followed by a definition of the limits, which vary depending on the rostro-caudal level (Figure9, Figure10, Figure11).

Figure 8 :

 8 Figure 8: localization of the locus coeruleus (LC)The reference slice was located at the rostral tip of the motor trigeminal nucleus (Mo5N). The LC was located at the ventro-lateral corner of the 4th ventricle (4V), just ventral to the mesencephalic trigeminal nucleus (Me5), delineated from the 4V by the central gray of the mesencephalon (CGPn). The LC had the aspect of a juxtaposition of white and black dots, resulting from an artifact produced by its neuromelanin content. This artifact was especially clear on T2-weighted 100µm. At lower resolution the LC appeared as a black area which is smaller. It had the shape of an ovoid having its main axis pointing supero-medially. ctg: centro-tegmental tract; MBP: medial parabrachial n.; PnO: oral part of the pontine reticular formation; scp: superior cerebellar peduncle. A, B: respectively axial and coronal slices, T2 weighted 100 µm. C, D: same segmented slices.

Figure 9 :

 9 Figure 9: rostral limit of the Locus coeruleusFrom the reference slice, the artifact was followed on coronal and sagittal views to localize its rostral limit, at the mid rostrocaudal level of the inferior colliculus (CI). At this level, the LC was located:• between the periaqueductal gray (PAG), ventro-medially, and the oral part of the pontine nucleus (PnO) ventrolaterally; • it was just ventro-medial to the mesencephalic trigeminal n. (Me5). A, B: respectively axial and coronal slices, T2 weighted 100 µm. C, D: same segmented slices.

Figure 10 :

 10 Figure 10: limits of the locus coeruleus (LC) at the caudal pole of the inferior colliculus (CI). • ventrally: oral part of the pontine reticular nucleus (PnO); • medially: central gray of the rhombencephalon (CGPn); • dorsally: mesencephalic trigeminal n. (Me5) laterally, and trochlear nerve (4n) medially; • laterally: superior cerebellar peduncle (scp). More caudally (Figure 8), the medial parabrachial nucleus (MPB) was interposed between the locus LC and scp. A, B: axial and sagittal slices respectively, T2 weighted 100 µm. C, D: same segmented slices.

Figure 11 :

 11 Figure 11: caudal limit of the locus coeruleus (LC) The artifact was followed up to its caudal limit (red dotted line) on coronal and sagittal views. It was located at the level of the rostral 1/4th of the motor trigeminal nucleus (Mo5N). Me5: mesencephalic trigeminal n.; MBP: medial parabrachial n.; PnO: oral part of the pontine reticular formation; scp: superior cerebellar peduncle; VLTg: ventro-lateral tegmental area; 4V: 4 th ventricle. A, B: axial and sagittal slices respectively, T2 weighted 100 µm. C, D: same segmented slices.

Figure 12 :

 12 Figure 12: segmentation of the facial nucleus (7N).CED/T2 185µm maps were used for rough location of 7N: an axial slice running just rostral to the pontomedullary sulcus, through the center of the abducens nucleus (6N) was selected. The facial nerve (7n) appeared as a black linear structure with a ventromedial curve; 7N was round and medial to the 7n. It appeared violet-blue on CED map and was delineated from the 6N by a blue darker area (intermediate reticular zone, IRt). Then, the limits of 7N were defined:• cranial and caudal: IRt;

  of 7n and more laterally, spinal trigeminal nucleus (Sp5); • medial: gigantocellular n. (Gi) in which fibers of the abducent nerve (6n) are running. A, B, C: respectively axial, coronal and sagittal slices, T2 weighted 100 µm. D, E, F: same slices ; overlay of T2 weighted 100 µm (100% opacity) and CED map (50% transparency). G, H, I: same segmented slices.

  .fr/brainstems/index.html). Depending on the user internet connection speed, the first download of the 3D objects may require a few seconds to 1 minute. Subsequent downloads are considerably faster thanks to the browser cache.

	WIKIBrainStem	is	hosted	by	Tours	University	(http://fibratlas.univ-
							Original	page	available	at:
						https://fibratlas.univ-
						tours.fr/mediawiki/index.php/Red_nucleus

Each page gives the following information: A, anatomical details; B, used datasets; C, segmentation rules; D, difficulties; E, 3D model. tours

Table 2 : Anatomical classes hierarchical list.

 2

Acknowledgements

We express our gratitude to the donor involved in the body donation program of the Association

Conversely, nuclei surrounded by white matter tracts were, indeed, much easier to segment thanks to an enhanced contrast between tracts (hyposignal) and nuclei (hypersignal). An initial delineation of such nuclei was usually performed on the diffusion-weighted quantitative DTI maps (ADC, FA, CED) before being refined on high-resolution anatomical T2-weighted maps (100µm). Diffusion sequences alone were, indeed, not sufficient to properly delineate nuclei, because some of them were crossed by, or contained, a large population of fibers; if an isodirectional population of fibers was connected with or arose from a nucleus, the FA increased at the level of the junction, and the fibers/nucleus contrast locally decreased. This was, for instance, the case for the posterior part of the facial nerve. Similarly, since FA reflects the overall directionality inside a voxel, it descended in areas where fibers crossed; if the resulting low-FA area was located close to a nucleus (also having a low FA value), the latter was artificially extended. In these cases, we were obligated to define arbitrary -but tentatively reproduciblerules that, of course, remain debatable. Future experiments will consist of investigating the potential of HARDI models to better disentangle such complex fiber configurations, but this is out of the scope of the current study.

Any segmentation protocol based on anatomical rules indeed contains a certain degree of arbitrariness and aims at providing a consensual and reproducible, if not perfect, parcellation scheme. An ideal validation for a dataset of smaller size would classically include multiple segmentations performed by different users of various levels of expertise and computation of similarity indices. Such a process seemed unrealistic for a 500-hour-long segmentation, since it would test a mixture of rule reliability and procedure tediousness. For this reason, the initial segmentation was performed by a single operator and reviewed by two senior authors who finally provided consensus-based segmentation and rules. A direct comparison of the segmentation performed on MR data to histological or OCT or PLI data on the same specimen would also be of great interest. Nevertheless, this has probably to be more regarded as the


Bernard Pierre Dufour

Wayan Kerana

Fabienne Ribeyre

Population dynamics of Hypothenemus hampei (Ferrari) according to the phenology of Coffea arabica L. in equatorial conditions of North Sumatra

Keywords: coffee tree, fruiting, climate, coffee berry borer, dispersal, Toba Highands

In the Toba Highlands of North Sumatra (Indonesia), coffee production (Coffea arabica L. var. Sigarar Utang) is an important outcome for smallholders; however, the attack of the coffee berry borer (CBB) Hypothenemus hampei, is an obstacle for the development of coffee cultivation in this area. This pest causes great economic losses produced by the development of its offspring inside the coffee berries, making it difficult to control. This concerning situation has led us to consider the development of a CBB control strategy, but beforehand, it was necessary to acquire key information on the phenology of the coffee tree and its implication on the bioecology of the pest. Thus, two study designs were set up, one comprising six plots with two different age classes and the other corresponding to a single plot dedicated only to the study of short distance dispersal of CBB. Part of this study focused on the phenology of the coffee trees and showed that berry production mainly takes place in the upper parts of the trees and significantly decreases with tree age. Due to the equatorial climate, berries were practically always present. Berries were produced following two major flowering periods and some minor ones distributed over the year, and harvested at regular intervals. Berry distribution on the branches varied over time.

Dynamics of infestations by CBB showed that ripe berries were more infested than unripe berries because they had been exposed longer to CBB attacks, that older trees were more exposed than younger trees and that infestation was evenly distributed along branches. In addition, internode pedestrian dispersal of CBBs was shown to occur, but considerably less frequently than airborne dispersal. In conclusion, it appears that in the agro-climatic context of the Toba region, the virtual year-round presence of berries -which fosters CBB infestations and CBB shortdistance dispersal -is a constraint that must be taken into consideration for designing

Introduction

The Sumatra archipelago is the main coffee-producing area in Indonesia. Its geographical diversity makes it possible to grow robusta coffee trees (Coffea canephora Pierre var. robusta) in the lowlands and Arabian coffee trees (Coffea arabica L.) on the high-elevation plateaus, in particular in the province of North Sumatra.

In the Simalungun district of North Sumatra, the dominant C. arabica variety is Sigarar Utang, mainly cultivated unshaded, around Lake Toba. This coffee, originating from a natural cross between the Tim Tim cultivar and a lineage of the Bourbon variety, belongs to the internationally-famous Mandheling Coffee group [START_REF] Mawardi | Geographic coffees from Indonesia and its potential to support world expresso coffee industry[END_REF][START_REF] Mawardi | Establishment of Geographical Indication Protection system in Indonesia, case in coffee[END_REF]. The Indonesian Coffee and Cocoa Research Institute (ICCRI) introduced this variety in the 1980s as an alternative to robusta cultivation. It is higher yielding than the Sumatra typical variety, which was once grown in the area, but it is described as moderately resistant to leaf rust caused by Hemilea vastatrix Berk. & Br and susceptible to the coffee berry borer (CBB) Hypothenemus hampei (Ferrari) Coleoptera: Curculionidae: Scolytinae), the major coffee pest worldwide [START_REF] Hulupi | Participatory breeding on Arabica coffee to obtain superior local varieties to support origin specialty coffee product development in Indonesia[END_REF]. Since the introduction of C. arabica in this district, productivity has remained fairly low, at approximately 50 or 65% only of its full potential [START_REF] Saragih | Socioeconomic and ecological dimension of certified and conventional arabica coffee production in North Sumatra, Indonesia[END_REF]. According to [START_REF] Saragih | Aspek sosioekologis usahatani kopi arabika di dataran tinggi kabupaten Simalungun, Sumatera Utara[END_REF], all coffee plantations are grown by smallholders and are said to be poorly managed due to lack of training and to agroecological shortcomings [START_REF] Dufour | Effect of coffee tree pruning on berry production and coffee berry borer infestation in the Toba Highlands (North Sumatra)[END_REF]. This unfavourable context for high productivity is made even worse by damage caused by the CBB reported by several authors [START_REF] Mawardi | Recent status of Coffee Berry Borer in Indonesia[END_REF][START_REF] Saragih | Socioeconomic and ecological dimension of certified and conventional arabica coffee production in North Sumatra, Indonesia[END_REF]. Indeed, by digging galleries inside the coffee beans to complete its life cycle, the CBB makes them unfit for consumption [START_REF] Vega | The genus Hypothenemus, with emphasis on H. hampei, the coffee berry borer[END_REF]. Thus, before being marketed, coffee must be sorted in order to eliminate CBB-damaged beans that are among the defective forms to be discarded.

In Indonesia, CBB was first detected in Java in 1909 [START_REF] Cramer | Review of literature of coffee research in Indonesia[END_REF] then in Sumatra in 1918 [START_REF] Corporaal | De koffieboorder op Sumatra's Oostkust en Atjeh[END_REF] before spreading to other islands and colonising all the coffee production areas. More recently, significant attacks by CBB were reported on C. arabica in North Sumatra. Monthly data from the district of Simalungun from May 2018 to September 2019 revealed a high percentage of holed berries, ranging from 32 to 58% (I W. Kerana, unpublished).

Face of this concerning sanitary situation, it was considered crucial to develop an efficient and sustainable strategy to control the pest and curb the infestation to acceptable levels in order to reduce harvest losses and shorten sorting operations, which heavily affect post-harvest costs. A pre-requisite to tackle this challenge was the acquisition of key information on the phenology of the coffee trees in the highelevation equatorial climate of the Lake Toba area, and on the impact of this phenology on the bioecology of the CBB.

It is precisely because the variety Sigarar Utang is a local arabica that it was important to study its fruit set and describe how it varies according to the trees' age.

In a large proportion of the plantations, the trees are left to develop freely during several years, which results in them gaining substantial height and crown volume. It was also essential to obtain data on the phenology of the cultivar Sigarar Utang, since the local climate conditions influence flowering and fruiting dynamics, and at this moment, they have never been studied. In this particular agro-climatic context, we also studied how the CBBs colonise the berries throughout the annual fruiting cycle, which is an important basis for developing an IPM program. We also studied the dynamics of infestation in relation to the age of the coffee trees, as in the case of a heavy infestation of older coffee trees, possible rejuvenation through pruning operations could bring benefits to IPM [START_REF] Dufour | Effect of coffee tree pruning on berry production and coffee berry borer infestation in the Toba Highlands (North Sumatra)[END_REF]. Moreover, for a more theoretical purpose, we studied the distribution of CBB attacks in each tree in order to identify possible preferences. In addition, given that flight seemed to be the only means of dispersal documented so far (Roman-Ruiz et al., 2018) we also examined the possibility of walking movement of CBBs between axillary nodes.

Material and methods

Description of the study sites

This work was carried out from March 2012 to April 2014 in the highlands of the Simalungun district, near Lake Toba (North Sumatra, Indonesia), at N 2°49' and E 98°50'. The six smallholder's plantations of C. arabica var. Sigarar Utang coffee selected for the study were located at an elevation of 1,200 m above sea level, close to three villages a few kilometres apart belonging to two different subdistricts (Pematang Sidamanik and Dolok Pardamean). The plantations were on flat ground, homogeneous and fully exposed to sunlight, and the trees were planted at 2 x 2 to 2 x 2.5 m densities. There were usually intercrops of red pepper, ginger or maize between rows. Plantation management consisted in applications of compost and chemical fertilisers three times a year for the intercrops and coffee trees. Weeding was carried out manually or chemically two or three times a year in all the plots. The trees were not pruned as in most plantations in the region, no CBB control means was used, and ripe berries were harvested at regular intervals of two to three weeks even in periods of very low production. Harvest was carried out by ourselves on the trees monitored in our study. Mean annual production of green coffee in the area reached 1,139 kg/ha [START_REF] Saragih | Produksi kopi arabika spesialti Sumatera Utara: análisis sosial ekonomo; ekologie da, keijakan pemerintah daerah[END_REF]. The design consisted in six 0.5-1 ha plots of unpruned coffee trees. The first year, it comprised two 4-5-year-old plots and four 6-8-year-old plots, but the following year while the young plots were retained, the oldest were replaced by 5-year-old plots. In each of these plots, 15 coffee trees were selected according to the same sampling design, i.e. 5 coffee trees per row, 10 m apart on 3 rows spaced 20 m apart.

On each tree, six productive branches were selected, either young primaries of the top of the tree or young secondaries emerging from older branches. The first year, we selected two opposite branches in the upper third of the tree, two in the middle and two in the lower third. The second year, we focused on the upper third of the trees only, selecting two branches on each of three levels within this upper third. This specific arrangement was designed to select branches bearing a greater number of nodes. Overall, according to this design, we selected a total of 6 x 15 x 6 = 540 branches, on which all our observations were carried out during the two years. The During each survey repeated every 21 days, every branch was visually inspected beginning at its insertion on the trunk and working towards its tip, counting the number of infested and intact berries at each node encountered, one node having at least one berry. Thus, all deeply perforated berries within the perimeter of the apical disc were considered infested by H. hampei because this disc is its preferred puncture site because of its rough surface [START_REF] Gumier-Costa | Por que fêmeas da broca do café perfuram preferencialmente a coroa dos frutos?[END_REF]. This species is monophagous, morphologically similar to other species of the same genus, however infested with other crops [START_REF] Vega | The genus Hypothenemus, with emphasis on H. hampei, the coffee berry borer[END_REF]. Only H. obscurus, usually polyphagous could lead to confusion, but its presence on coffee is occasional [START_REF] Constantino | Aspectos biológicos, morfológicos y genéticos de Hypothenemus obscurus e Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae)[END_REF]. Berries were classified into two categories: 'large unripe' (green, 5 mm or more in diameter) and 'ripe' (entirely red). Berries under 5 mm in diameter were not counted. The 5 mm size corresponds roughly to the stage at which the berries begin to become palatable to CBBs and suitable for puncturing (Salazar-Gutierrez et al., 1993). Mid-ripe berries were included in the 'large unripe' category.

All ripe berries were harvested after each survey, throughout the two study periods.

Design for Hypothenemus hampei (Coleoptera: Curculionidae) short-range dispersal study

The experimental design was set up in May 2013 near the village of Manik Saribu (Simalungun district) at N 2° 49" 8"' and N 98° 50" 35'", in a single plot of 0.5 ha infested by CBBs. The objective was to assess the H. hampei' potential to disperse by walking along the branches. We selected six primary, productive, branches on each of ten unpruned coffee trees inside the study plot. On each of these branches, we circled the branch with glue bands (Pelton Glu®, France) between each axillary node, as well as before the first and after the last node (Fig. 2).

Starting the day the bands were positioned, green, mid-ripe and ripe berries in each node were counted every two weeks over 20 weeks, distinguishing between intact and infested berries and ripe berries were harvested after each survey.The CBBs caught on the glue traps between the nodes were also counted. Females are characterised by their size, which varies from 1.5 to 1.9 mm, about 1/3 larger than the males [START_REF] Roepke | Gegevens omtrent de Koffiebessen-boeboek (Stephanoderes hampeï Ferr. = coffeae Hgd[END_REF][START_REF] Corbett | Some preliminary observations on the coffee berry borer, Stephanoderes (Cryphalus) hampei Ferr[END_REF][START_REF] Bergamin | Contribuição para o conhecimento da biologia da broca do café Hypothenemus hampei (Ferrari, 1867)(Col. Ipidae)[END_REF][START_REF] Vega | The genus Hypothenemus, with emphasis on H. hampei, the coffee berry borer[END_REF].

According to the literature, females are always more abundant than the males but in varying proportions and the males have atrophied wings and never leave the berries [START_REF] Hargreaves | Notes on the coffee Berry borer (Stephanoderes hampei, Ferr.) in Uganda[END_REF][START_REF] Corbett | Some preliminary observations on the coffee berry borer, Stephanoderes (Cryphalus) hampei Ferr[END_REF][START_REF] Pelley | Pests of coffee[END_REF][START_REF] Damon | A review of biology and control of the coffee berry borer, Hypothenemus hampei (Coleoptera: Scolytidae)[END_REF][START_REF] Vega | The genus Hypothenemus, with emphasis on H. hampei, the coffee berry borer[END_REF][START_REF] Mariño | Sun vs. shade affects infestation, total population and sex ratio of the coffee berry borer (Hypothenemus hampei) in Puerto Rico[END_REF]. In this trial, the identification of some captured beetles was confirmed in the laboratory (CIRAD/France), in particular with the use of reference specimens of H. hampei.

Data analysis

For the multi-site study, we took into account the position of the axillary nodes on the fruiting branches (fruiting zone) by dividing each branch into three segments of subequal length: the segment closest to the trunk (branch base), the central segment, and the end segment (branch tip). Moreover, the coffee trees were divided into two age classes: 5 years and under, and 6 years and above, because differences in plant height, branch size and foliage volume could cause variations in berry production and CBB infestations.

About the phenology of Coffea arabica L. var. Sigarar Utang, the distribution of the counted berries per segment of the fruiting zone of each branch was analysed at the beginning of both years of study, soon after the main flowering in February. A negative binomial distribution was fitted to the data for each of the two tree-age classes using chi-squared minimisation and tested with the chi-square goodness-offit test.

The effect of the age of coffee trees, and the combined effect of branch level in the tree and tree age, on the production of ripe berries over the period were assessed with the data of Year 1 using two non-parametric Kruskal-Wallis rank-sum tests. Dunn's test for multiple comparisons was used to compare the levels of the variable combining the three branch levels and the two tree-age classes, with Benjamini-Hochberg's adjustment to control the experiment-wise error rate.

The fruiting dynamics was analysed in Year 1 and Year 2 on branches of the upper third only (the most productive) of the coffee trees. The variations of the total number of berries and of the proportion of infested berries were analysed in the three segments of the fruiting zone of each branch, for each tree and tree-age class. The total number of berries was assumed to vary over time, increasing after flowering and decreasing with each harvest. The proportion of infested berries was assumed to vary according to the available resource (berries) and the development conditions of the CBB population. Thus, the two were expected to have a complex non-linear relationship with time. To model this, we used a generalised additive model with mixed effects (GAMM), which incorporates nonlinear dependence. We used it with a negative binomial distribution for berries and a binomial distribution for the number of infested berries in relation to the number of intact berries. The GAMM models integrated a fixed effect of age, season and position, two smooth terms based on time, and a random effect to take into consideration the correlation between different days on the same coffee tree. The smooth terms used a thin plate spline with dependence on position for one and on age for the other to take into account the general dynamics and the differential due to position and age.

Statistical analyses were performed using R version 3.6.1 (R core Team, 2019).

The fitting of negative binomial distributions was carried out using the R package vcd [START_REF] Meyer | Visualizing Categorical Data[END_REF]). Dunn's test for multiple comparisons was performed using the R packages FSA [START_REF] Ogle | FSA: Fisheries Stock Analysis[END_REF] and rcompanion [START_REF] Mangiafico | rcompanion: Functions to Support Extension Education Program Evaluation[END_REF]. The GAMM models were implemented using the R packages mgcv [START_REF] Wood | Generalized additive models: an introduction with R (2 nd edition)[END_REF] and itsadug (Van Rij et al., 2017). Graphs were plotted using the R package ggplot2 [START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF].

Results

Climate of the Lake Toba area

The climate of the area of Lake Toba is among the most typically equatorial climates (Encyclopaedia Universalis, 2020). According to the Köppen classification system [START_REF] Peel | Updated world map of the Köppen-Geiger climate classification[END_REF], this climate corresponds to the "Cfa" class (without dry season and hot summer). The annual amplitude of temperature is very narrow and precipitations are frequent practically all year round (Fig. 3). Given that the elevation is close to 1,200 m above sea level, mean temperature is around 21°C, with a tenday temperature amplitude rarely exceeding 10°C. Relative humidity is usually high at night but the ten-day average is approximately around 80%. Over the study period, we observed that the ten-day rainfall was quite uniformly distributed except in June and July 2012 and 2013 and in January to February 2014, when precipitations abated. Overall, these variations are in keeping with the pattern of the mean monthly precipitations recorded from 2000 to 2011 (Fig. 1). The total annual rainfall recorded in 2012 and 2013 was respectively 2,533 mm and 2,643 mm.

Phenological characteristics of Coffea arabica, var. Sigarar Utang

In the climatic conditions that prevailed locally, four flowering periods were observed annually from 2012 through to 2014 (Fig. 4). During the main major bloom, the statistical distribution of the number of berries per segment of the fruiting zone was overdispersed. Negative binomial distributions could be fitted to the data from both young 4-5-year-old coffee trees and older 6-8 year-old trees in Year 1 (Xsquared = 87.0, df = 74, p-value = 0.143, and X-squared = 65.9, df = 52, p-value = 0.093, respectively), but only to the data from the older coffee trees in Year 2 (Xsquared = 52.8, df = 64, p-value = 0.841). In Year 1, a large number of the axillary nodes of the fruiting zone (73% and 79% of nodes in younger and older trees respectively) failed to bear berries, but 10% of the nodes in the younger trees and 1.75% in the older trees numbered 10 berries or more. In Year 2, these age-related differences were less marked, with respectively 70% and 72% of the axillary nodes of the fruiting zone bearing no fruit, while 8% of nodes in both age groups bore 10 berries or more.

Berry production in relation to tree age and branch level

Figure 5 shows that in Year 1 the younger coffee trees were significantly more productive than the older (with a mean of 119 ripe berries on six branches versus 52; Kruskal-Wallis test, p < 0.001).

The Kruskal-Wallis test also detected a significant difference between ageclasses and branch location in the tree (p < 0.001). Applied after the significant Kruskal-Wallis test, Dunn's multiple comparison test distinguished three classes of ripe berry production (Fig. 5). The more productive locations are the highest (67) and intermediate (50) branches of young coffee trees, followed by, in decreasing order, the higher branches of the older trees (36), the lower branches of young trees (17), and the intermediate ( 15) and finally lower branches of the older trees (6), these being the least productive of all. In spite of four flowering periods every year contributing to berry production, both in Year 1 and Year 2 the total quantity of berries present on branches followed a gradually decreasing trend from March-April to February of the following year (Fig. 6).

The first bloom was thus, quantitatively, the most productive. However, the four annual blooms resulted in the presence practically all year round of unripe, ripening and completely ripe berries -all palatable and attractive to CBBs.

In Year 1 and Year 2 alike, berries from flowers fertilised during the mid-February bloom (Table 1) started to reach the 'large unripe' stage in March-April (Fig. 6). Taking as reference the production of Year 2, which was greater, the mean number of berries per set of six branches increased from 117 to 126 between April and May due to the duration of the preceding flowering period. Thereafter, from May to December of the same year, the number of berries declined continuously, despite the input of young berries resulting from the second-main bloom of August and the minor blooms of May and November (Table 1, Fig. 6). These inputs can be perceived on Fig. 6, in particular on 23 August and 15 November 2013. The number of berries, large unripe and ripe taken together, reached its yearly low on 21 March 2014.

In contrast to what is seen in large unripe berries, the number of ripe berries does not increment with each passing bloom because they are regularly harvested (Fig. 6). At each harvest, their number remains fairly stable. Overall, ripe berries are present all year round but reach their annual low between February and April.

Production dynamics of Coffee arabica berries on high branches according to tree age and node position

Term significance in the GAMM confirmed that higher branches are more productive in young trees than in older trees (Table 2). The smooth curve of ripe berries dynamics based on predictions from the model showed that production decreased more slowly over the year in young trees than in older trees (Fig. 7A), with the latter catching up a little on this difference after some 350 days. Regarding the distribution of berries over the three segments of the fruiting zone, their number decreased from branch base to branch tip (Table 2). In the fruiting zone of upper branches, however, the dynamics observed differed depending on the segment, with an inversion of the general pattern in the course of the production period as branch tips became the more productive segment (Fig. 7B). The smooth curve of the ripe berries dynamics on the branches of all coffee trees, thus showed that production decreased regularly. In the beginning, production was significantly higher close to the trunk than in the middle of the branch, where it was greater than at the tip. The distribution of ripe berries along the branches later evened out in the middle of the period, and reversed before the end (Fig. 7B). The model explained 43% of the deviance (Table 1).

Parametric coefficients

Esti Term significance in the GAMM confirmed that the proportion of infested berries was generally higher on the old coffee trees monitored in Year 1 (Table 3). The smooth curve of the infestation dynamics based on predictions from the model showed that the infestation rate increased evenly until around day 340 -with a significant age-related difference that narrowed down around day 280 -then declined as the harvest was drawing to a close (Fig. 8A). The proportion of infested berries remained fairly stable whatever the position on the branch (Table 3). The smooth curve of the infested berries dynamics showed that their proportion increased gradually until around day 350 with only slight position-related differences, after which the infestations remained significantly higher closer to the trunk (Fig. 8B). The model explained 48% of the deviance (Table 2).

Parametric coefficients

Dispersal of H. hampei on branches

Twice monthly observations of the glue-traps on either side of the 300 axillary nodes monitored in this study showed that the first female CBBs began to appear in the traps after the first infested berries were recorded (Fig. 9). All trapped CBBs were females. Numbers of trapped CBBs never exceeded 14 individuals per observation date, and dwindled to zero as soon as no infested ripe berry remained. A total of 47 female CBBs were trapped. This number must be put into perspective because these captured females were close to nodes that have borne 335 infested ripe berries over a period of four and a half months, which implied the emergence and then flight of hundreds of other females. It should be noted that a single infested berry may harbour several dozen adults and larval stages from the oviposition [START_REF] Vega | The genus Hypothenemus, with emphasis on H. hampei, the coffee berry borer[END_REF] and that more than three generations may occur [START_REF] Baker | The coffee berry borer in Colombia. Final report of the DFID-CENICAFE-CABI Bioscience IPM for coffee project (CNTR 93/1536A)[END_REF].

Discussion

In the field, the Sigarar Utang variety, regarded by [START_REF] Hulupi | Participatory breeding on Arabica coffee to obtain superior local varieties to support origin specialty coffee product development in Indonesia[END_REF] as highly productive, was characterised by an uneven production at all levels -cluster, branch and tree. A majority of nodes bore no berry at all, while some 8% comprised more than 10. Berry production was significantly higher in young trees of 4-5 years than in older trees of 6-8 years (Fig. 5). In both cases, it was more abundant in the upper sections of the crown than in the lower parts (Fig. 5). According to Coste (1969), branches of the upper storeys are short young primary branches, more abundantly supplied in sap and therefore capable of producing more flowers and fruits. Conversely, the more meager production of the lower, older, branches can be explained by a faltering supply of sap; this situation then degrades into a completely unproductive stage, followed by a progressive drying out. A recent study in the area of Lake Toba showed that pruning these low branches produces a regenerative effect that triggers the development of secondary and tertiary shoots bearing fertile nodes, and stimulates fruiting [START_REF] Dufour | Effect of coffee tree pruning on berry production and coffee berry borer infestation in the Toba Highlands (North Sumatra)[END_REF]. It can thus be stated that pruning the coffee trees can offset their age-related decline in productivity.

Concerning the dynamics of berry production and development, we show that the succession of blooms over the year result in the presence of berries on the branches practically at all times. This flowering and fruiting pattern, already mentioned in [START_REF] Dufour | Effect of coffee tree pruning on berry production and coffee berry borer infestation in the Toba Highlands (North Sumatra)[END_REF], is also observed in certain areas of Colombia between the 4 th and 5 th degrees of latitude North at high elevations [START_REF] Arcilla-Pulgarín | La floración del cafeto y su relación con el control de la broca[END_REF]. This particular phenology is related to the prevalence of a long period of frequent rains interrupted by a few short dry spells (which, in the Simalungun area, mostly take place in the beginning and middle of the year). Arcilla-Pulgarín and Jamarillo-Robledo ( 2003) and [START_REF] Arcila-P | Floración del café en Colombia y su relación con la disponibilidad hídrica, térmica y de brillo solar[END_REF] showed that in equatorial regions where the photoperiod is under 13 hours, the flowering of coffee trees is narrowly linked to water deficits or to high daily variations of the temperature.

In contrast to the productive pattern observed in tropical regions of Western Africa [START_REF] Borbón-Martínez | Bioécologie d'un ravageur des baies de caféier, Hypothenemus hampei Ferr. (Coleoptera: Scolytidae) et de ses parasitoïdes au Togo[END_REF], Mexico and Central America [START_REF] Barrera | Dynamique des populations du scolyte des fruits de caféier, Hypothenemus hampei (Coleoptera: Scolytidae), et lutte biologique avec le parasitoïde Cephalonomia Stephanoderis (Hymenoptera: Bethylidae), au Chiapas, Mexique[END_REF], which

allows a single annual harvest only, in the Simalungun district, the staggered production combined with the frequent harvest of ripe berries results in the progressive decline of the total number of berries between April-May and February.

This was observed in all coffee trees, and in particular in branches heavily loaded with fruits of the upper third of the trees (Fig. 6). It follows that berries can be found on branches over a period of at least ten months out of twelve, thus giving H. hampei a constant supply of food and reproduction sites next to its places of emergence.

In order to study the production pattern in greater detail, we focused on the dynamics of berry production and development in relation to the age of the trees and the position of berries on the branches. Our analyses confirm that young coffee trees were more productive than older trees (Fig. 7A) and show that at the beginning of the harvesting season berries were more numerous at the base of the branches, near the trunk, than at the tip, whereas the situation reversed towards the end of the season (Fig. 8b).

In this remarkable phenological configuration in which all the development stages of berries co-occur during most of the year, we could observe that the proportion of CBB-infested berries was more than twice as high among ripe berries (mean: 46%) as among unripe berries (mean: 18%). This trend is explained by the fact that ripe berries had been exposed to CBB attacks for longer than unripe berries, and that their potential to attract CBBs was greater [START_REF] Giordanengo | Evidence for allelochemical attraction of coffee berry borer, Hypothenemus hampei, by coffee berries[END_REF][START_REF] Mendoza | Atractividade de frutos a broca-do-café, Hypothenemus hampei (Ferrari): estímulos visuais e olfativos[END_REF]. This difference in attractiveness could be due to the diversity of the volatile organic compounds produced and emitted by ripe berries [START_REF] Ortiz | Volatile composition of coffee berries at different stages of ripeness and their possible attraction to the coffee berry borer Hypothenemus hampei Coleoptera: Curculionidae)[END_REF].

Regarding the infestation dynamics, our results show that the proportion of perforated berries perceived as infested, as the number of berries was decreasing with each successive harvest (Fig. 8A and B, Fig. 7A). However, this dynamic is probably overestimated if we consider that some perforated berries, described here as infested, may have been abandoned by the colonizing females before they reach the endosperm [START_REF] Ruiz-Cárdenas | Life table of Hypothenemus hampei (Ferrari) in relation to coffee berry phenology under Colombian field conditions[END_REF]. We also show that aged coffee trees were always more infested than younger trees, even though this difference narrowed down during the later part of the harvest season, when the number of berries generated by the first major bloom had substantially decreased on aged trees (Fig. 8 and Fig. 7). On the other hand, the distribution of infested berries on individual branches remained fairly similar at different levels in the tree throughout the harvest season despite the variations in fruiting dynamics mentioned earlier. The cooccurrence of older, infested, berries with younger, intact, berries explains this uniform distribution of infestations. In tropical settings, where coffee trees produce a single large annual crop, colonisation tends to spread gradually, aggregatively, and to become homogeneous when infestation levels are high [START_REF] Román-Ruiz | Short-distance dispersal of Hypothenemus hampei (Ferrari) females (Coleoptera: Curculionidae: Scolytinae) during the coffee tree fruiting period[END_REF].

The dispersal of female CBBs by flight was often considered as an intangible principle, in particular in the context of the large-scale migrations that motivated the development and implementation of trapping techniques in tropical regions [START_REF] Gutiérrez-Martínez | Trampeo en el campo, de la broca del fruto de café Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) con los semioquímicos volátiles del fruto de café Robusta Coffea canephora Pierre ex Froehner[END_REF][START_REF] Dufour | Optimization of coffee berry borer, Hypothenemus hampei Ferrari (Col. Scolytidae), mass trapping with an attractant mixture[END_REF]. However, we here demonstrate for the first time that CBBs also disperse by walking. Even though this dispersal is on a small scale, it nonetheless contributes to the progressive infestation of the axillary nodes of a branch (Fig. 9). According to our observations, dispersal by pedestrian locomotion concerns females only (as males spend their entire life cycle inside the infested berry) and occurs in the immediate vicinity of the infested ripe berry from which they emerged. This type of movement, not observed in tropical areas [START_REF] Román-Ruiz | Short-distance dispersal of Hypothenemus hampei (Ferrari) females (Coleoptera: Curculionidae: Scolytinae) during the coffee tree fruiting period[END_REF], reinforces the idea that, in the equatorial zone, dispersal would more often concern short distances than in tropical areas [START_REF] Dufour | Effect of coffee tree pruning on berry production and coffee berry borer infestation in the Toba Highlands (North Sumatra)[END_REF], at the scale of the individual branch or tree. Future experimental trapping at the scale of the plot will undoubtedly yield new elements for furthering the discussion on dispersal.

Conclusion

In this study undertaken in a high-elevation equatorial climate setting of the Lake Toba area in North Sumatra, we presented the phenological characteristics of the arabica var. Sigarar Utang coffee tree and their relationships with CBB infestations.

We showed: (i) how the staggered fruiting covering most of the year allowed the practically uninterrupted development of CBB infestations despite the frequently repeated harvest of ripe berries; (ii) that the older coffee trees were more affected than the younger trees; (iii) and that the horizontal distribution of infestations on individual branches remained fairly stable over time. In the ideal trophic environment that are coffee trees for CBB development, females tend to disperse by flight in search of hosts, but mainly at short range, within the tree where they emergedwhich would a priori make this pest more difficult to control, in particular through trapping means. Trapping operations target above all females emerging from residual berries fallen on the ground, and only becomes effective in tropical areas at the time of the migration peaks that take place after the harvest, outside the fruiting period [START_REF] Dufour | Piégeage de masse du scolyte du café Hypothenemus hampei Ferr[END_REF]. The trapping trials we are considering will probably not produce the same result in number of captures, but their efficacy is still unknown. Our opinion however is that sanitation harvesting, recommended in many countries and by a number of agronomists advocating non-chemical control [START_REF] Decazy | Le scolyte du fruit du caféier, Hypothenemus hampei: considérations sur la lutte intégrée contre ce ravageur[END_REF][START_REF] Bustillo-Pardey | Una revisión sobre la broca del café, Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae) en Colombia[END_REF][START_REF] Aristizábal | Integrated pest management of coffee berry borer: strategies from Latin America that could be useful for coffee farmers in Hawaii[END_REF] could become a key tool for CBB management because it would primarily concern infested berries still on the trees, during the fruiting period. We would also recommend completing this approach with some pruning of the coffee trees, which would directly facilitate sanitation harvesting interventions [START_REF] Dufour | Effect of coffee tree pruning on berry production and coffee berry borer infestation in the Toba Highlands (North Sumatra)[END_REF], and with measures to prevent reinfestation by dispersing female CBBs during post-harvest operations. It will probably be necessary to identify other components of control to design a strategy for integrated CBB management in the region of Lake Toba.

  Temperature and humidity were measured and recorded at the study site with a Testo 175 data logger at 2-hour intervals. Rainfall data were supplied by the Tea Research and Development Center PTPN IV -Tobasari-Pematang Sidamanik. The average rainfall data from 2000 to 2011 used as a reference were provided by the same Centre (Fig 1), but no such average temperature and relative humidity were available. 2.2. Design for fruiting characteristics of Coffea arabica L. var. Sigarar Utang and observations about Hypothenemus hampei (Coleoptera: Curculionidae) infestation

  periods spanning from March 2012 to February 2013 and from April 2013 to April 2014 are referred to as Year 1 and Year 2 respectively.

3. 4 .

 4 General dynamics of Coffee arabica var. Sigarar Utang production and berry development in Years 1 and 2

Figure 1 .

 1 Figure 1. Average monthly rainfall in the Toba region for the period 2000-2011

Figure 4 .

 4 Figure 4. Annual flowering periods of coffee trees in all experimental plots. Dark colored bars correspond to major blooms (main and second main) and light colored bars correspond to minor blooms. Small flowering bursts sometimes occur on a few trees outside the periods indicated; they are not included in the figure.

Figure 5 .

 5 Figure 5. Cumulated ripe berry production of two branches in Year 1 according to the age of the coffee trees and the position of the branches in the tree. The thick line of the boxes-and-whisker plots represents the median. The upper and lower edges of the boxes represent the upper and lower quartiles. The whiskers represent 1.5 times the interquartile range.

Figure 6 .

 6 Figure 6. Production dynamics of coffee berries in Years 1 and 2 according to their stage of maturity. Each rectangle represents the average number of berries per set of six branches at a given date.

Figure 8 .

 8 Figure 8. Variation in the proportion of infested berries on coffee trees over the period according to tree age (A) and position on branch (B). Smoothing is based on predictions from a generalised additive mixed model that included the effects of position on branch (base, middle or tip), tree age and year, two smooth terms based on the number of days (time), with dependence on position and age, and a coffee tree random effect.

Figure 9 .

 9 Figure 9. Number of H. hampei females trapped next to axillary nodes in relation to the dynamics of infestation of coffee tree branches.

Table 1 .

 1 Results of generalised additive mixed models (GAMM) of berries dynamics.Over 540 branches monitored, a mean of 15.9% and 19.6% of the large unripe berries were infested in Year 1 and Year 2 respectively, and 38.8% and 53.8% of the ripe berries, with ripe berries systematically harvested every three weeks.

	-mate	Std. Error *	z-value	p-value

Acknowledgements

The authors would like to thank Edouard Bault, Mélanie Landthaler, Yuliana Dolok Saribu and Laurent Bossolasco for their assistance in the local organisation of our research activities, and Anya Cokkle-Bétian for translating the manuscript into English. They also wish to express their gratitude to the coffee producers of the Lake Toba area for their unwavering collaboration, as well as to CIRAD and the French Embassy in Indonesia for their financial support.