Julien Pansiot
email: julien.pansiot@inria.fr

Lionel Reveret
email: lionel.reveret@inria.fr

Edmond Boyer
email: edmond.boyer@inria.fr

Combined Visible and X-Ray 3D Imaging

Keywords: motion capture, tomography, x-ray, colour video

This paper considers 3D imaging of moving objects and introduces a technique that exploits visible and x-ray images to recover dense 3D models. While recent methods such as tomography from cone-beam x-ray can advantageously replace more expensive and higher-dose CT scanners, they still require specific equipment and immobilised patients. We investigate an alternative strategy that combines a single x-ray source and a set of colour cameras to capture rigidly moving samples. The colour cameras allow for coarse marklerless motion tracking, which is further refined with the x-ray information.

Once the sample poses are correctly estimated, a dense 3D attenuation model is reconstructed from the set of x-ray frames. Preliminary results on simulated data compared to ground-truth as well as actual in-vivo experiments are presented.

Ground truth

Reconstruction Skin isosurface Skeleton isosurface (256 slices) (from 32 views) Figure 1: Rat paw volumetric reconstruction from a sparse set of simulated visible and xray images (32 frames from a rotational motion). Left-to-right: ground truth CT volume, volumetric reconstruction from 32 views rendered as planar x-ray, isosurface for soft tissues interface, and skeleton interface.

Introduction and related work

The capture of movements to study functional analysis has gained great attention in the recent years with the improvement of acquisition systems. In that respect, the ability to capture simultaneously the motion of both internal structures such as the skeleton and external surfaces is a challenge with promising applications. Whilst a range of solutions are available c 2014. The copyright of this document resides with its authors. It may be distributed unchanged freely in print or electronic forms.

for motion capture and analysis in 3 dimensions using visual cues, most are solely limited to the recovery of surface information. Conversely, radiography allows for the capture of inner structures in motion, but is mostly restricted to 2 dimensions. In this paper we proceed one step towards full volumetric 3D motion capture by investigating the case of rigidly moving samples, relying on a combination of visible and x-ray cameras, and assuming no prior knowledge on the captured sample.

Our aim is to recover dense 3D models of moving objects. The existing two main strategies for radiography are the regular planar x-ray and the Computed Tomography (CT) scanner. While the latter produces high-resolution dense 3D models, it suffers three main drawbacks: higher radiation dose for the patient [START_REF] Siewerdsen | Volume CT with a flat-panel detector on a mobile, isocentric C-arm: pre-clinical investigation in guidance of minimally invasive surgery[END_REF], higher cost and, more importantly here, immobility of the patient during the procedure. For these reasons, specific procedures have been developed to generate volumetric attenuation models from a limited number of regular cone-beam x-ray sources, such as full 3D models from isocentric/orbital C-arm [START_REF] Siewerdsen | Volume CT with a flat-panel detector on a mobile, isocentric C-arm: pre-clinical investigation in guidance of minimally invasive surgery[END_REF], and increasingly well resolved models for breast tomosynthesis [START_REF] Yang | A nonlinear least squares method for solving the joint reconstruction and registration problem in digital breast tomosynthesis[END_REF]. Model-based methods using bi-planar x-ray beams [START_REF] Benameur | A hierarchical statistical modeling approach for the unsupervised 3-D biplanar reconstruction of the scoliotic spine[END_REF] even allow motion capture [START_REF] Brainerd | Xray reconstruction of moving morphology (XROMM): precision, accuracy and applications in comparative biomechanics research[END_REF], however such methods require strong prior models and usually manual intervention.

In order to compute a dense 3D attenuation model while limiting the patient dose, allowing for motion capture and incidentally reducing cost, we propose a novel approach that combines regular colour cameras with standard x-ray imagery to acquire a rigidly but freely moving markerless sample. Our framework keeps all equipment static, eliminating the need for specific isocentric C-arms or other tightly controlled systems [START_REF] Siewerdsen | Volume CT with a flat-panel detector on a mobile, isocentric C-arm: pre-clinical investigation in guidance of minimally invasive surgery[END_REF]. The approach does not use prior anatomic models either, hence this is neither a registration problem [START_REF] Yang | A nonlinear least squares method for solving the joint reconstruction and registration problem in digital breast tomosynthesis[END_REF], nor a model fitting issue [START_REF] Benameur | A hierarchical statistical modeling approach for the unsupervised 3-D biplanar reconstruction of the scoliotic spine[END_REF] and it allows for unknown shapes. The paradigm we follow here does not consider motion as noise but, on the contrary, as one key to 3D reconstruction.

The closest approach to ours has been proposed by Sidky et al. [START_REF] Sidky | Accurate image reconstruction from few-views and limited-angle data in divergent-beam ct[END_REF] where a reconstruction method for any type of calibrated x-ray imaging device under the assumption of limited angles and/or number of views is presented. Schumacher et al. [START_REF] Schumacher | Combined reconstruction and motion correction in SPECT imaging[END_REF] also proposed a method that combines reconstruction and motion correction in SPECT imaging, without requiring colour cameras. This method is however tailored for motion correction and hence appears to cater only for a limited range of motion. The use of colour video for motion detection during e.g. SPECT has been covered for example by [START_REF] Mcnamara | A flexible multicamera visual-tracking system for detecting and correcting motion-induced artifacts in cardiac SPECT slices[END_REF], but requires markers. A markerless method has been proposed by [START_REF] Hutton | A hybrid 3-D reconstruction/registration algorithm for correction of head motion in emission tomography[END_REF], but is again targeted to small motion correction.

To the best of our knowledge, our approach is the first to use motion, through the combination of visible and x-ray images, to perform 3D radiography. Potential medical applications include affordable dental 3D imaging, 3D imaging in confined environments, and, when non-rigidities will be handled, joints inner dynamics.

The remainder of this paper is composed of the following: in Section 2 we describe our reconstruction method, in Section 3 we present our results from both simulated and in-vivo data, before concluding in Section 4.

Proposed method

The proposed method relies on a single x-ray source combined with a set of regular colour cameras. The colour cameras are used to build a 3D surface model that is tracked over time. The motion estimation of this model is refined using the x-ray images. Once the motion is known, a dense 3D attenuation model can be reconstructed from the x-ray data. The overall pipeline is illustrated in Figure 2.

Calibration

Both visible and x-ray cameras are fully calibrated in world coordinate system prior to acquisition. This operation is performed in two stages to minimise the x-ray usage. Firstly, the eight video cameras are fully calibrated (intrinsic and pose) on their own, using a moving LED wand within a single bundle adjustment framework. Secondly, a lightly coloured lead ball is displaced within the entire capture volume, triangulated by the visible cameras, before the x-ray camera is finally fully calibrated knowing the 3D from calibrated video cameras.

Initial pose estimation

Since the sample can move freely within the capture volume, it is necessary to estimate its pose prior to reconstruction. Using the colour cameras only, the silhouettes of the sample segmented from the background are processed with a polyhedral visual hull algorithm [START_REF] Franco | Exact polyhedral visual hulls[END_REF] to generate a 3D mesh of the sample per frame (see fig. 2). The remaining meshes of the sequence are then registered to a reference pose model, e.g. the first frame, with a robust Iterative Closest Point (ICP) implementation with outlier detection. This provides an initial pose, i.e. rotation and translation, estimation for each frame and with respect to the reference frame.

Pose refinement

Since the visual hull meshes exhibit artefacts due to the limited number of colour cameras, the initial pose estimation is corrupted. To improve the pose, we exploit directly the x-ray silhouettes, segmented from the x-ray background light.

To this aim, we rely on the assumption that if the poses were perfectly registered, the volume reprojection on the x-ray plane should equate perfectly to the x-ray silhouettes. For simplicity, we consider the model to be static and captured by moving cameras, with intrinsic parameters from the initial calibration, and extrinsic from the initial pose estimation. A cost function penalising differences between the original x-ray silhouettes and the reprojected model is used within a gradient descent framework to refine the poses iteratively. If C is the set of N camera parameters, I the set of N segmented images, M a mesh, 'proj' the camera projection operator, and 'vh' the visual hull operator, we try to estimate:

arg min C N ∑ i=1 |proj C i (M) -I i | st. M = vh C (I). (1)
This extra stage also reduces slight spatial and temporal misalignment between the visible and x-ray systems, providing more robust results.

Reconstruction

Once the pose is known, the Kaczmarz method [START_REF] Kaczmarz | Angenäherte auflösung von systemen linearer gleichungen[END_REF], also referred to as Algebraic Reconstruction Technique (ART) is used to reconstruct iteratively the volumetric attenuation. For enhanced performance, the views are ordered such that two successive points of view are most orthogonal, and the reconstruction takes places only within the x-ray visual hull.

Given the volume X being reconstructed, the observed images A with a saturated attenuation A sat , and a relaxation parameter λ i , we exploit the projection matrix sparseness by precomputing each camera ray defined as the list of the N intersected voxels in the reconstructed volume p and their associated weights w. For each ray R i = {(p i,1 , w i,1 ), ..., (p i,N , w i,N )} associated to the image pixel value A i , we then compute the norm, the current integrated attenuation on the ray x i , and the weighted difference with the observed pixel value, d i :

R i 2 = N ∑ j=1 w 2 i, j , x i = N ∑ j=1 w i, j X [p i, j ] , d i = λ i A i -x i R i 2 . (2)
The voxel attenuations are then updated as follows:

X n+1 [p i, j ] =      0 if X n [p i, j ] + d i w i, j < 0 (positivity constraint), X n [p i, j ]
if d i < 0 and A i = A sat (prevent saturation artefacts), X n [p i, j ] + d i w i, j in other cases.

(

The relaxation parameter λ i is weighted for each pose based on the leave-one-out score calculated as in eq. ( 1) in order to favour poses which appear more reliably estimated.

Since the amount and nature of the observed data may render the problem locally illposed, high-frequency noise is observed in the results. Using the assumption that living organisms can be modeled by a set of relatively homogeneous tissues, a 3D adaptation of Rudin et al. seminal non-linear Total Variation (TV) [START_REF] Rudin | Nonlinear total variation based noise removal algorithms[END_REF] is enforced on the model in between iterations for regularisation, as proposed by [START_REF] Sidky | Accurate image reconstruction from few-views and limited-angle data in divergent-beam ct[END_REF]. This improves dramatically the results, as illustrated in Figure 3.

Experiments and results

Simulated data

The combined x-ray and visible images can be simulated by rendering real volumetric CT data using raycasting. In this experiment, we rendered the CT model of a rat paw (256 3 voxels), simulating a system of eight colour cameras and one x-ray device. The combined set of 2D images was then processed through the whole pipeline. The effective projection resolution for both systems was 600×600 pixels. The model was artificially moved continuously for 32 frames, following roughly a 180-degree rotation. This allowed for comparison with ground-truth data. Figures 1 and3 show the original model as well as the reconstructed volume.

In-vivo experiment

A system composed of eight colour cameras (2048×2048 pixels) and one Siemens AR-CADIS Avantic x-ray C-arm (1024×1024 pixels) was deployed for this preliminary experiment. The reconstruction volume (256 3 voxels) covered a cube of 20×20×20 cm, ie. a resolution of 0.78mm/voxel. Three volunteers (one of them shown here) were asked to perform roughly a 180-degree wrist circumduction with fingers kept as rigid as possible within the working volume, during which 32 frames were captured. The colour cameras recorded at 30 fps, whilst the x-ray acquired at 10 fps to minimise the dose, both being only coarsely synchronised. Figure 4 illustrates the preliminary results of the proposed method. Left-to-right: volume rendered as planar x-ray, soft tissues thresholding, skeleton thresholding, skeleton thresholding without pose refinement: some phalanges are thicker, the distal noisier. The soft tissues exhibit a concavity that cannot be modelled solely by visual hulls.

It is evident from the results that whilst the simulated data demonstrates the proposed approach's great potential, the preliminary reconstruction of in-vivo data is noisier. We attribute this difference to the fast shutter noise in x-ray images (in particular in the metacarpal area) as well as non strictly rigid motion (on the finger tips and the thumb in particular). The former should be improved by carefully planned x-ray acquisition parameters.

Conclusion and future work

In this paper we have shown that a multi-camera system can advantageously complement a single static x-ray imaging device and enable 3D motion imaging of rigidly moving samples. Future work includes the analysis of the achievable quality based on the recorded motion and non-rigid motion handling using rigid-by-part modelling.

Figure 2 :

 2 Figure 2: Overview of the proposed pipeline for combined visible and x-ray 3D imaging.

Figure 3 :

 3 Figure 3: Two rat paw reconstructed slices from simulated visible and x-ray images (32 frames). Left-to-right: ground-truth CT data (256 slices), motion estimated with ICP and TV regularisation, motion refined but without TV, motion refined and TV, true motion and TV. Slight motion estimation errors are clearly corrected by the refinement stage, but not as well as with true motion. TV regularisation reduces significantly the high-frequency noise.

Figure 4 :

 4 Figure 4: Human hand reconstruction with a combined visible & x-ray system (32 frames).Left-to-right: volume rendered as planar x-ray, soft tissues thresholding, skeleton thresholding, skeleton thresholding without pose refinement: some phalanges are thicker, the distal noisier. The soft tissues exhibit a concavity that cannot be modelled solely by visual hulls.

Acknowledgments

This project was funded by KINOVIS (ANR-11-EQPX-0024) and MORPHO (ANR-10-BLAN-0206), with help from Amiqual4Home (ANR-11-EQPX-0002).


Alain Dejean
email: alain.dejean@wanadoo.fr

Messika Revel

Frédéric Azémar

Olivier Roux

Sven Thatje

Altruism during predation in an assassin bug

Keywords: Conspecific tolerance, Predation, Prey sharing, Reduviidae, Zelus annulosus

Zelus annulosus is an assassin bug species mostly noted on Hirtella physophora, a myrmecophyte specifically associated with the ant Allomerus decemarticulatus known to build traps on host tree twigs to ambush insect preys. The Z. annulosus females lay egg clutches protected by a sticky substance. To avoid being trapped, the first three instars of nymphs remain grouped in a clutch beneath the leaves on which they hatched, yet from time to time, they climb onto the upper side to group ambush preys. Long-distance prey detection permits these bugs to capture flying or jumping insects that alight on their leaves. Like some other Zelus species, the sticky substance of the sundew setae on their forelegs aids in prey capture. Group ambushing permits early instars to capture insects that they then share or not depending on prey size and the hunger of the successful nymphs. Fourth and fifth instars, with greater needs, rather ambush solitarily on different host tree leaves, but attract siblings to share large preys. Communal feeding permits faster prey consumption, enabling small nymphs to return sooner to the shelter of their leaves. By improving the regularity of feeding for each nymph, it likely regulates nymphal development, synchronizing molting and subsequently limiting cannibalism.

Introduction

Group hunting followed by food sharing brings fitness benefits to the participants through increased per capita food intake while minimizing the cost of capture. Indeed, in addition to avoiding predation, preys are captured more effectively with less risk of injury. This is known in social spiders [START_REF] Settepani | Task specialization in two social spiders, Stegodyphus sarasinorum (Eresidae) and Anelosimus eximius (Theridiidae)[END_REF][START_REF] Yip | The influence of siblings on body condition in a social spider: is prey sharing cooperation or competition?[END_REF] and some ant species [START_REF] Cerda | Predation in the Hymenoptera: an evolutionary perspective[END_REF]) and, among vertebrates, in fishes [START_REF] De Waal | Fishy cooperation[END_REF], birds, cetaceans, otters, carnivorans, and chimpanzees (see the synthesis in [START_REF] Bailey | Group hunting within the Carnivora: physiological, cognitive and environmental influences on strategy and cooperation[END_REF]. Harris hawks cooperatively hunt and rear young [START_REF] Coulson | Group hunting by Harris' hawks in Texas[END_REF]. Hyenas, Lycaon , wolves, and lions also redistribute food between adults [START_REF] Bailey | Group hunting within the Carnivora: physiological, cognitive and environmental influences on strategy and cooperation[END_REF], as do eusocial insects.

Delayed benefits from food sharing occur in vampire bats which regularly fail to obtain blood, their only food, and die after 70 h if starved. Unsuccessful individuals can receive regurgitations from roost-mates, frequently relatives. Yet, receiving regurgitations depends mostly on having previously provided the helper with blood, resulting in the formation of food-sharing groups characterized by mutual allogrooming [START_REF] Carter | Food sharing in vampire bats: reciprocal help predicts donations more than relatedness or harassment[END_REF]. A chimpanzee possessing food, particularly meat, can share it with close relatives or in response to harassment by beggars or obtain a delayed benefit through grooming and forming coalitions according to dominance [START_REF] Silk | Chimpanzees share food for many reasons: the role of kinship, reciprocity, social bonds and harassment on food transfers[END_REF]. Altruistic motivation for food sharing exists in bonobos which share their food with a stranger rather than with a group mate, but do not give food if a social interaction is impossible [START_REF] Tan | Bonobos share with strangers[END_REF].

In insects, egg clutching, in addition to improving egg survival by preventing desiccation, is often associated with toxicity or impalatability and favors larval/nymphal aggregation [START_REF] Janz | Evolutionary ecology of oviposition strategies[END_REF] which frequently permits early instars to organize a group defense [START_REF] Jolivet | ) Encyclopedia of entomology[END_REF]). Feeding efficiency is improved when larval aggregation is associated with communal feeding in the herbivore larvae of sawflies, chrysomelid beetles, and lepidopterans [START_REF] Wilson | The insect societies[END_REF][START_REF] Grégoire | Larval gregariousness in the Chrysomelidae[END_REF]). Communal feeding is infrequent among predatory insects that lay their eggs in clutches. Group hunting and prey sharing were noted in the first instar larvae of aphidophagous ladybirds [START_REF] Moore | Does social feeding improve larval survival of the two-spotted lady beetle, Adalia bipunctata?[END_REF], in the nymphs and adults of a predacious pentatomid (Heteroptera) [START_REF] Ables | Notes on the biology of the predacious pentatomid Euthyrhynchus floridanus (L.)[END_REF], and in some harpactorine assassin bug nymphs (Heteroptera; Reduviidae) [START_REF] Inoue | Studies on the mode of foraging of the gregarious assassin bug Agriosphodrus dohrni Signoret[END_REF][START_REF] Haridass | Predatory behavior of Rhynocoris marginatus Fabricius (Harpactorinae-Reduviidae-Heteroptera-Insecta)[END_REF][START_REF] Jackson | Specialized prey selection behavior of two East African assassin bugs, Scipinnia repax and Nagusta sp. that prey on social jumping spiders[END_REF].

Except for the vertebrate blood-feeding Triatominae, other reduviid subfamilies are almost exclusively composed of arthropod predators [START_REF] Hwang | Evolutionary history of assassin bugs (Insecta: Hemiptera: Reduviidae): insights from divergence dating and ancestral state reconstruction[END_REF]; but see [START_REF] Bérenger | Relations privilégiées de certains Heteroptera Reduviidae prédateur avec les végétaux. Premier cas connu d'un Harpactorinae phytophage[END_REF]. Prey capture success in most harpactorine species, related to prey size [START_REF] Inoue | Group predatory behavior by the assassin bug Agriosphodrus dohrni Signoret (Hemiptera: Reduviidae)[END_REF][START_REF] Cogni | Influence of prey size on predation success by Zelus longipes L. (Het., Reduviidae)[END_REF], is enhanced by the presence of a sticky substance that coats their legs. "Resin bugs" gather this substance from certain plants [START_REF] Forero | Resin gathering in neotropical resin bugs (Insecta: Hemiptera: Reduviidae): functional and comparative morphology[END_REF]. In some species of the genus Zelus, the substance is secreted by specialized dermal glands and sticks to the "sundew setae" (so-called because they resemble the trichomes of sundew carnivorous plants) that cover the tibiae of the forelegs [START_REF] Werner | Surface morphology of legs in the assassin bug Zelus longipes (Hemiptera: Reduviidae): a scanning electron microscopy study with an emphasis on hairs and pores[END_REF][START_REF] Betz | The role of adhesion in prey capture and predator defence in arthropods[END_REF][START_REF] Weirauch | Observations on the sticky trap predator Zelus luridus Stål (Heteroptera, Reduviidae, Harpactorinae), with the description of a novel gland associated with the female genitalia[END_REF][START_REF] Zhang | Sticky predators: a comparative study of sticky glands in harpactorine assassin bugs (Insecta: Hemiptera: Reduviidae)[END_REF]. Because they lack these sticky glands, the first instars use the sticky secretions deposited by females on their egg mass to increase their predation success [START_REF] Weirauch | Observations on the sticky trap predator Zelus luridus Stål (Heteroptera, Reduviidae, Harpactorinae), with the description of a novel gland associated with the female genitalia[END_REF][START_REF] Law | Sticky substance on eggs improves predation success and substrate adhesion in newly hatched Zelus renardii (Hemiptera: Reduviidae) instars[END_REF]. Also, Agriosphorus dohrni nymphs (Harpactorinae) allocate short periods to active foraging where they form search groups of ten or more individuals of different origins (not necessarily siblings); small preys are eaten selfishly, whereas large preys are shared [START_REF] Inoue | Studies on the mode of foraging of the gregarious assassin bug Agriosphodrus dohrni Signoret[END_REF][START_REF] Inoue | Group predatory behavior by the assassin bug Agriosphodrus dohrni Signoret (Hemiptera: Reduviidae)[END_REF].

The nymphs of the assassin bug Zelus annulosus are efficacious predators that develop mostly on Hirtella physophora (Chrysobalanaceae) [START_REF] Revel | An assassin among predators: the relationship between plant-ants, their host myrmecophytes and the Reduviidae Zelus annulosus[END_REF]. This is an understorey-dwelling myrmecophyte (i.e., a plant housing ants in hollow structures) whose long-lived leaves bear extrafloral nectaries and a pair of pouches at the base of each lamina where colonies of the myrmicine ant, Allomerus decemarticulatus , shelter. Workers of this species build gallery-shaped traps permitting them to capture preys 1,800 times their weight [START_REF] Dejean | Arboreal ants build a trap to ambush and capture prey[END_REF][START_REF] Dejean | Predation success by a plant-ant indirectly favours the growth and fitness of its host myrmecophyte[END_REF]Grangier et al. 2008a). Allomerus workers tolerate the fragile Z. annulosus first instar nymphs on their host plants (these nymphs are thus protected from other ants) and, later, the relatively large last instar nymphs do not attack these ants [START_REF] Revel | An assassin among predators: the relationship between plant-ants, their host myrmecophytes and the Reduviidae Zelus annulosus[END_REF].

Although several authors have provided information on the predatory behavior of different harpactorine species [START_REF] Edwards | Observations on the life history and predatory behaviour of Zelus exsanguis (Stål) (Heteroptera: Reduviidae)[END_REF][START_REF] Inoue | Studies on the mode of foraging of the gregarious assassin bug Agriosphodrus dohrni Signoret[END_REF][START_REF] Inoue | Group predatory behavior by the assassin bug Agriosphodrus dohrni Signoret (Hemiptera: Reduviidae)[END_REF][START_REF] Haridass | Feeding and behavioural parameters and egg ultrastructure in the biosystematic of Reduviidae[END_REF][START_REF] Haridass | Predatory behavior of Rhynocoris marginatus Fabricius (Harpactorinae-Reduviidae-Heteroptera-Insecta)[END_REF][START_REF] Weirauch | Zelus renardii and Z. tetracanthus (Hemiptera: Reduviidae): biological attributes and the potential for dispersal in two assassin bug species[END_REF][START_REF] Zhang | Sticky predators: a comparative study of sticky glands in harpactorine assassin bugs (Insecta: Hemiptera: Reduviidae)[END_REF], we aimed to verify if clutch laying by females is followed by nymph clustering with the subsequent possibility of group hunting and prey sharing by examining the modalities of their hunting behavior.

Materials and methods

Study site and models

This study was conducted between 2009 and 2013 at the field station at Petit Saut, Sinnamary, French Guiana (5°03′39″ N; 53°02′36″ W) and in the forest around the station; also, some Z. annulosus individuals were gathered from La Montagne des singes (5°04′19″ N; 52°41′42″ W), ca. 50 km away from Petit Saut. The climate is tropical moist, with 3,500 mm of annual precipitation distributed over 280 days.

Z. annulosus was only found on pubescent plants, mostly H . physophora. The females lay egg clusters under the leaves and deposit a sticky secretion on them that seems sufficient to ward off predators and ants. They then fly away, leaving the eggs to develop unattended. Between 10 and 22 first instar nymphs hatch from these egg clusters [START_REF] Revel | An assassin among predators: the relationship between plant-ants, their host myrmecophytes and the Reduviidae Zelus annulosus[END_REF]AD, MR, and OR, personal communication).

The hunting zones and preys of different instars of Z. annulosus nymphs

We surveyed 65 Hirtella trees on which we had noted the presence of Z. annulosus nymphs, representing ca. 240 h of focal samplings distributed over 80 non-consecutive days during the rainy season in which individuals were observed continuously for periods of time of more than 2 h (generally limited by the rain or nightfall). In addition, during a series of observations permitting us to cover the 24 h of the nycthemeron, we verified each hour if the nymphs were hunting or sheltering under the leaves (35 to 43 diurnal controls; 16 to 48 nocturnal controls).

In both approaches, we noted if, depending on the instar, the nymphs stayed under the leaf where they hatched or left to hunt on its upper side, whether they remained grouped or not, and finally if they moved to a different leaf. We also noted what insects were captured by these nymphs plus information on their size and the period of the year. Using a quartz crystal microbalance, we weighed several live preys and Z . annulosus nymphs to calculate the prey-predator weight ratio (Table 1).

Prey capture behavior by Z. annulosus nymphs

This study was conducted on nymphs bred in the laboratory in transparent plastic boxes (180×120×75 mm) whose floor was covered with humid blotting paper. Each nymph, starved for 72 h after being placed in the plastic box, was tested only once. The protocol was adapted from that used for solitary foraging ants (see [START_REF] Dejean | Prey capture behavior in an arboreal African ponerine ant[END_REF]. During preliminary experiments, a full repertoire of behavioral sequences was first established. Referring to this complete list, the prey capture behavior was studied in the laboratory using third (N =56) and fifth (N =28) instar Z. annulosus nymphs confronted with termite workers (Nasutitermes nigriceps) which are easy to find and rather uniform in size. After a prey was dropped from ca. 50 mm in height, we recorded the complete sequence of behavioral acts performed by the nymphs until they abandoned the remains of the prey. We then built flow diagrams where the transition frequencies between behavioral acts, presented as percentages, were calculated based on the overall number of cases.

Role of the sundew setae during the capture of flying and jumping insects

We tested third instar nymphs bred in the same laboratory conditions as previously but using larger transparent plastic boxes (240×180×100 mm) inside which we installed a paper shelf in one corner of the box, 20 mm below the cover. Two sets of 14 nymphs were starved for 72 h before one set was provided with 27-30-mm-long adult Tipulidae and the other with 10-12-mm-long grasshoppers. Each nymph was tested only once. The tested preys were first placed in a refrigerator at 4 °C during 30 min before being very gently deposited on the paper shelf in the plastic boxes so as to keep them from being immediately detected. They remained immobile during 5-6 min before leaving the paper shelf and were captured when they happened to walk close to the ambushing nymphs. We noted the behavioral sequences.

Prey sharing by Z. annulosus nymphs: influence of prey size, predator size, and degree of hunger We verified if first instar nymphs, although fragile in appearance, are able to capture and share preys. Using smooth forceps, we gathered nine groups of nymphs and placed each group in a transparent plastic box and transported them to the laboratory. After starving them for 24 h and still using smooth forceps, we deposited them on the upper side of a horizontal H. physophora leaf placed in the plastic boxes. After 5 min, the nymphs apparently adapted to this situation as they did not immediately move under the leaves and remained grouped. We dropped a termite from ca. 50 mm in height onto the center of the leaf and noted if the nymphs captured and shared the prey.

We tested the influence of the degree of starvation and the size of the preys in a series of experiments based on groups of five or six second instar nymphs. We bred them in transparent plastic boxes (240×180×100 mm) where we placed an old, resistant Hirtella leaf (they have an acumen; see Grangier et al. 2008a) maintained 30 mm above the floor, thanks to a piece of modeling clay. Also, a small test tube containing cotton imbibed with water served as a watering place. After being starved for 96 h, 14 groups of nymphs were provided with one termite worker; we verified if the nymphs shared the prey. After the same period of starvation, 14 other groups of nymphs were provided with 12-14-mm-long katydids and here, too, we verified if the preys were shared; 36 h later, we provided these 14 groups (they were not yet food deprived as they had eaten a katydid 36 h before) with a termite worker and again noted if the preys were shared or not.

Further observations were made in the forest to verify if the behaviors differed from those noted in the laboratory. We also Late instar nymphs fan out over different Hirtella leaves to ambush (MR, personal communication). Because we noted that they shared preys, we hypothesized that a successful nymph can attract tree-mates. Due to the horizontal position of Hirtella leaves, it was easy to eliminate the visual cues that can attract individuals ambushing on lower leaves by placing a prey on one of the upper leaves. We very gently placed a 12-14-mm-long numbed (by freezing) katydid on the uppermost leaf of each surveyed Hirtella tree while fourth or fifth instar nymphs were ambushing on lower leaves and noted the number of nymphs present on that leaf after 30 min of feeding on the katydid. We compared two situations (25 cases each): absence (control) versus presence (trial) of an ambushing nymph on the upper leaf when we deposited the katydid.

In 22 cases similar to the previous control (here control 1), we noted the length of time separating the depositing of the katydid from the arrival of a first nymph. For 11 of these cases, we waited until a second nymph arrived (trial 1); for the 11 other cases, we removed the first nymph prior to it attacking the katydid (control 2). In both cases, we noted the length of time separating the arrival of the first and second nymphs. These data were compared with 15 cases where a nymph was ambushing on the upper leaf, noting the length of time separating the depositing of the katydid from the arrival of a "second" nymph (trial 2).

In a third experiment, conducted on 16 Hirtella trees sheltering last instar Zelus nymphs, we deposited two katydids on two lower leaves while a nymph was ambushing on one of them. We compared the length of time separating the depositing of the katydids from the arrival of a nymph on each of the two leaves. Statistical analyses were conducted using GraphPad Prism 5.02 software.

Results

The hunting zones and preys of different instars of Z. annulosus nymphs

In the field, the first instar nymphs remained grouped under the leaf where they hatched until the first molt and only exceptionally climbed up onto the upper side of that leaf where they all sat and waited, their forelegs upward, ambushing in a group (noted once for eight groups monitored hourly between 8:00 and 18:00 hours during six nonconsecutive days). The second and third instar nymphs devoted much more time to group ambushing on the upper side of their leaves (noted 28 times for the eight groups monitored during 12 non-consecutive days, the two instars pooled; comparison with the previous case, Fisher's exact test: P <0.001). The rest of the day and at night, nymphs of all stages remain grouped under the protection of their leaves; hunting nymphs immediately sheltered under the leaves when the rain began (27 cases noted). We observed that less than 5 min separated the moment when the first and the last individual climbed up onto the upper side of their leaf to ambush in a group. On the contrary, diurnally, the fourth and fifth instar nymphs mostly ambushed solitarily on different leaves as they are large enough to avoid the traps built by Allomerus ants as they slowly move from leaf to leaf. This was the case for 47 observations out of 96 (12 series of observations of eight groups, the two instars pooled; 48.9 %), but twice they were grouped on a leaf (2.1 %; but see below).

Because they ambush on the leaves of their host trees, Z. annulosus nymphs captured flying insects almost uniquely, the exceptions being jumping Orthoptera nymphs (Table 2). The second instar nymphs captured only small (i.e., less than 8 mm), fragile preys (i.e., Aleyrodidae and different Diptera; likely underrepresented in Table 2), while larger instars attacked almost all insects that tried to land on their leaf, including items larger than themselves. Seasonality was noted for certain preys as all horse flies were captured in October (when they abound during the dry season), winged termites were captured after the first rains in December and January and winged ants between mid-December and March.

The Z. annulosus nymphs can feed on the prey captured by the Allomerus on their trap (cleptobiosis was noted nine times) or on flies, social wasps, and stingless bees attracted by these captured preys.

Prey capture behavior by Z. annulosus nymphs (laboratory studies)

The predatory sequences developed by small and large Z . annulosus nymphs were quite similar although the predatorprey weight ratios were different (see Table 1). When ambushing, nymphs generally lift and spread their forelegs. Preys, detected from a distance by visual or chemical cues or vibrations, were cautiously approached; the nymphs lifting their forelegs higher than previously. When at an adequate distance, they slammed their forelegs down on the preys stuck on the sundew setae; then, the nymphs extended their rostrum toward the preys to bite them in an inter-segmental zone, paralyzing them. The Z . annulosus lifted the preys by extending their rostrum and later used their forelegs to rotate the preys while injecting saliva and sucking out digested matter; the corresponding parts of the preys turned black. The Z . annulosus nymphs can repeatedly rotate and bite the preys up to ten times before discarding the emptied out preys (Figs. 1 and2).

Role of the sundew setae during the capture of flying and jumping insects (laboratory studies) Both tested preys were detected by sight immediately after they began to leave the paper shelf on which they had been deposited; indeed, the nymphs oriented their bodies in the preys' direction while lifting their antennae and forelegs. The attacks occurred immediately after the preys happened to approach the nymphs to less than 20 mm. All of the 14 tested Tipulidae were captured in flight on the first attempt as one or both of their long, thin wings became stuck to the nymphs' forelegs and they became paralyzed after being bitten. Among the 14 tested grasshoppers, 10 (71.4 %) were mastered on the first attempt because they lost their hind legs through autotomy at contact with the sundew setae of the Zelus nymphs' forelegs (see Fig. 2d). Among the four remaining grasshoppers, three were mastered on the first attempt although they did not lose their hind legs and the last one escaped. Here, too, the bite triggered immediate paralysis. As was noted for termites, after becoming paralyzed, both the Tipulidae and the katydids were rotated and bitten numerous times before being abandoned.

Prey sharing by Z. annulosus nymphs: influence of prey size, predator size, and degree of hunger (laboratory studies) All tested groups of starved first instar nymphs almost simultaneously attacked and then shared a termite prey.

Although the size between termite workers and katydids is not the only difference, the importance of prey size likely explains the difference noted when starved groups of second instar nymphs shared a termite worker in only 28.6 % of the cases, while they shared a much larger katydid in all cases. The effect of hunger was illustrated as 36 h after capturing a katydid as prey these "rather replete" nymphs this time shared a termite worker in 92.8 % of the cases (Fig. 3; see also prey sharing in natural conditions in Fig. 2).

When the captured termites were not shared, the nymph that successfully captured them moved away from its siblings. At the end of the sequence, it walked to the tip of the Hirtella leaf, placed its head above the extremity of the narrow acumen, and selfishly ate the termite. Indeed, none of the other nymphs tried to reach the prey from the underside of the leaf, and it was out of their reach from the upper side. The same was noted for five cases during controls conducted in the forest.

When testing if Z . annulosus nymphs from different geographic areas can form mixed groups, we noted that nymphs from all eight artificial groups remained grouped exactly as did siblings and that they similarly shared the tested preys.

Are nymphs attracted when preys are shared by late instar Z. annulosus nymphs?

From field studies, we have gathered arguments showing that Z. annulosus nymphs successful at capturing a prey likely Fig. 2 Capture of grasshoppers and katydids by Z. annulosus. a A small grasshopper was just seized and bitten by a fifth instar nymph. b A grasshopper is rotated and bitten in several parts. c A katydid nymph was just captured by a fifth instar nymph and had lost one hind leg in the process. d A small grasshopper, captured by an adult Zelus, had lost its hind legs through autotomy; the latter are still glued to the forelegs of the Zelus. The horizontal bars represent 10 mm. e Two nymphs sharing a fly. f An adult katydid was captured by a fifth instar nymph and is now being shared with three other nymphs, one of them biting this prey on the joint between the femur and the tibia of the hind leg Fig. 3 Prey sharing according to its size and the degree of hunger of second instar Z. annulosus nymphs (N =14 in each case). Statistical comparison: Fisher's exact tests; simultaneous tests were adjusted using the false discovery rate ("BY" correction as the P values are not independent; Pike 2011); different letters indicate significant differences at P <0.01 attract siblings; the tests being based on comparing nymphs attracted toward a prey alone (controls) to their attraction to a prey already having been captured by a sibling (trials). First, 30 min after a nymph had begun to feed on the numbed katydid provided, we noted significantly more nymphs on these leaves (trial) than on control leaves (2.64±0.21 vs. 0.36±0.11; N =25 in each case; Welch's corrected t test: t = 9.371; df =36; P <0.0001). Second, we did not note a significant difference between control 1 (length of time separating our depositing a numbed katydid and the arrival of a first nymph) and control 2 (when we removed the first nymph that reached the leaf where we had deposited the katydid) (Fig. 4). The length of time corresponding to these controls was significantly longer than that corresponding to trial 1 (i.e., separating the arrival of the two first nymphs) and trial 2 (i.e., an ambushing nymph was already present), while the difference between these latter situations was not significant (Fig. 4). Third, the length of time separating the depositing of katydids onto two lower leaves of 16 Hirtella trees to the arrival of a "new" last instar nymph was significantly shorter when a Zelus nymph was present (trials) than for the controls (25.5±1.9 vs. 14.3±0.9 min; paired t test: t =5.47; df= 15; P <0.0001).

Discussion

Although they are apparently fragile and never leave the underside of the H . physophora leaves where they hatched [START_REF] Revel | An assassin among predators: the relationship between plant-ants, their host myrmecophytes and the Reduviidae Zelus annulosus[END_REF] since they risk being trapped by Allomerus ants, we show that Z. annulosus first instar nymphs are able to capture and share comparatively large preys of up to 4.28 times their weight (Table 1). This capacity likely permits them to avoid lethal starvation which is recurrent in tropical species [START_REF] Maran | Paralytic potential of Catamiarus brevipennis (Serville), a potential biological control agent (Insecta: Heteroptera: Reduviidae)[END_REF]. In natural conditions, few flying insects of adequate size are prone to alight under the leaves of their host plant so that their main preys are adults of gallmaking Cecidomyiidae, which are frequent under the leaves of shrubs in the understorey (AD, personal observation).

The sequences of behavioral acts during prey capture by solitary nymphs were very similar to those known for other Harpactorinae, except that Z. annulosus ambush preys and detect them from a "long" distance, while, in other species, the nymphs forage actively and frequently detect the preys through antennal contact [START_REF] Edwards | Observations on the life history and predatory behaviour of Zelus exsanguis (Stål) (Heteroptera: Reduviidae)[END_REF][START_REF] Inoue | Studies on the mode of foraging of the gregarious assassin bug Agriosphodrus dohrni Signoret[END_REF][START_REF] Inoue | Group predatory behavior by the assassin bug Agriosphodrus dohrni Signoret (Hemiptera: Reduviidae)[END_REF][START_REF] Haridass | Feeding and behavioural parameters and egg ultrastructure in the biosystematic of Reduviidae[END_REF][START_REF] Haridass | Predatory behavior of Rhynocoris marginatus Fabricius (Harpactorinae-Reduviidae-Heteroptera-Insecta)[END_REF][START_REF] Jackson | Specialized prey selection behavior of two East African assassin bugs, Scipinnia repax and Nagusta sp. that prey on social jumping spiders[END_REF][START_REF] Weirauch | Zelus renardii and Z. tetracanthus (Hemiptera: Reduviidae): biological attributes and the potential for dispersal in two assassin bug species[END_REF]. Sundew setae are known to play an important role in the capture of caterpillars and small flying insects [START_REF] Weirauch | Zelus renardii and Z. tetracanthus (Hemiptera: Reduviidae): biological attributes and the potential for dispersal in two assassin bug species[END_REF][START_REF] Zhang | Sticky predators: a comparative study of sticky glands in harpactorine assassin bugs (Insecta: Hemiptera: Reduviidae)[END_REF]. Here, we show that they permit the capture of large insects prone to escape easily by flying or jumping away. Furthermore, contact with the sundew setae of the Zelus nymphs' forelegs triggered the autotomy of the hind legs of a grasshopper in 10 out of 14 cases. Autotomy, a mechanism designed to help an insect elude a predator's grasp, occurred here at the expense of the prey. Finally, in all cases, the injection of saliva was determinant due to its fast, non-lethal paralytic effect related to the presence of neurotoxic compounds (see [START_REF] Maran | Paralytic potential of Catamiarus brevipennis (Serville), a potential biological control agent (Insecta: Heteroptera: Reduviidae)[END_REF][START_REF] Corzo | Novel peptides from assassin bugs (Hemiptera: Reduviidae): isolation, chemical and biological characterization[END_REF][START_REF] Sahayaraj | Insecticidal activity of venomous saliva from Rhynocoris fuscipes (Reduviidae) against Spodoptera litura and Helicoverpa armigera by microinjection and oral administration[END_REF]. Communal nymphal feeding on large preys has been reported in several harpactorine species [START_REF] Inoue | Studies on the mode of foraging of the gregarious assassin bug Agriosphodrus dohrni Signoret[END_REF][START_REF] Haridass | Predatory behavior of Rhynocoris marginatus Fabricius (Harpactorinae-Reduviidae-Heteroptera-Insecta)[END_REF][START_REF] Jackson | Specialized prey selection behavior of two East African assassin bugs, Scipinnia repax and Nagusta sp. that prey on social jumping spiders[END_REF]. Although A. dohrni nymphs are active foragers while Z . annulosus nymphs are ambushers, in both species, small preys are eaten selfishly, whereas large preys are shared [START_REF] Inoue | Studies on the mode of foraging of the gregarious assassin bug Agriosphodrus dohrni Signoret[END_REF][START_REF] Inoue | Group predatory behavior by the assassin bug Agriosphodrus dohrni Signoret (Hemiptera: Reduviidae)[END_REF] this study). The groups of Z. annulosus nymphs are formed of siblings originating from the same egg clutches that ambush together and almost simultaneously attack large insects. So, peaceful food sharing may be related to kin selection [START_REF] Hamilton | The genetical evolution of social behavior. I, II[END_REF]). Yet, although they generally form groups of siblings in nature, we show that Z. annulosus nymphs tolerate non-kin conspecifics and even share preys with them as did A. dohrni nymphs in nature [START_REF] Inoue | Studies on the mode of foraging of the gregarious assassin bug Agriosphodrus dohrni Signoret[END_REF]. Note that the tolerance of conspecific aliens exists in the very similar situation of Allomerus plant-ants associated with H. physophora or Cordia nodosa, another myrmecophyte (Grangier et al. 2008b; see also papers cited therein).

It is probable that group living enhances protection from predators in early instar Z. annulosus nymphs as flying insects, mostly social wasps, regularly visit their host trees, trying to steal preys from the Allomerus ants [START_REF] Dejean | The hunter becomes the hunted: when cleptobiotic insects are captured by their target ants[END_REF]. This predation pressure explains why these nymphs stay underneath the leaves where they hatch. Because Z. annulosus nymphs remain immobile, insect predators may have trouble locating them when they are in the shade of their leaves and, because they remain grouped, they successfully counter-attack enemies. Communal feeding, which occurs for comparatively large insects (see prey-predator weight ratios in Table 2) plus small preys when the nymphs are replete, likely permits the effective exploitation of resources by pooling saliva and enabling a more rapid external digestion (see [START_REF] Schaefer | Prosorrhyncha (Heteroptera and Coleorrhyncha)[END_REF][START_REF] Forthman | Toxic associations: a review of the predatory behaviors of millipede assassin bugs (Hemiptera: Reduviidae: Ectrichodiinae)[END_REF]. The passage from selfishness to altruism through the "passive" sharing of small preys according to the degree of hunger of the nymphs, whereas large preys are shared, could regulate the food intake between nymphs, facilitating their synchronous development and molting. This limits size differences between the individuals of each group, a factor favoring cannibalism in assassin bugs, particularly for molting nymphs [START_REF] Inoue | Nymphal cannibalism in relation to oviposition behavior of adults in the assassin bug, Agriosphodrus dohrni Signoret[END_REF].

The main novelty of this study is the presence of "active" sharing for late instar nymphs that attract siblings to feed communally on large preys although they hunt solitarily on different leaves, but we do not know if this attraction is due to pheromones or vibrations, both being known in Hemiptera [START_REF] Kölliker | Maternal food provisioning in relation to condition-dependent offspring odours in burrower bugs (Sehirus cinctus)[END_REF][START_REF] Nomakuchi | Provisioning call by mothers of a subsocial shield bug[END_REF][START_REF] Kavčič | Tremulatory and abdomen vibration signals enable communication through air in the stink bug Euschistus heros[END_REF]. The fact that these nymphs ambush on different leaves and can attract siblings to share large preys increases the probability that each of them feeds regularly, while here, too, favoring the synchronization of the development and molting of the nymphs (see the similar size of the nymphs in Fig. 2f), limiting cannibalism. Because the preys are very attractive to predatory social wasps and stingless bees [START_REF] Dejean | The hunter becomes the hunted: when cleptobiotic insects are captured by their target ants[END_REF], the faster the siblings arrive, the less chance there is that a successful nymph will have its prey stolen or even be killed in turn. Moreover, arriving siblings can capture these cleptobionts (see Table 2) as do Allomerus ants [START_REF] Dejean | The hunter becomes the hunted: when cleptobiotic insects are captured by their target ants[END_REF]. Sharing large preys can result in a "by-product benefit" for the others (i.e., no cost for the donor, a benefit for the receiver; [START_REF] Leimar | By-product benefits, reciprocity, and pseudoreciprocity in mutualism[END_REF] or a "food for food" mutualism when a previous donor can later share a large prey captured by one of its siblings (see [START_REF] Stevens | A conceptual framework for nonkin food sharing: timing and currency of benefits[END_REF].

As a result, it is likely that communal feeding by Z . annulosus nymphs permits siblings to reduce the likelihood of starvation, decrease the time of exposure to predators, synchronize the development of siblings limiting the possibility of cannibalism while permitting the continuity of equitable mutual prey sharing, and finally increase their probability of reaching adulthood. The whole occurs through a selfsustaining process regulated by simple rules such as clutch laying triggering group living and ambushing by early instars (non-kins are tolerated), sharing a prey or not depending on its size and the degree of hunger of the successful nymph, and last instars attracting siblings to share large preys.

Fig. 1

 1 Fig. 1 Flow diagrams representing the sequences of the hunting behavior of Z. annulosus nymphs of two size classes during the capture of termite workers. The percentages were calculated from the total number of cases

Fig. 4

 4 Fig. 4 Comparison of the length of time (means ± SE) between (1) our depositing of a numbed katydid on the uppermost horizontal leaf of Hirtella trees and the arrival of the first Z. annulosus nymph (control 1 in the text), (2) the arrival of the second nymph (experiment 1), (3) the arrival of the second nymph, whereas we had removed the first nymph that reached the leaf where we had deposited the katydid (control 2), and

Table 1

 1 Information on the predator-prey weight ratio for the different cases studied. The weight of the Zelus annulosus nymphs and adults corresponds to an average for five to ten individuals starved for 4 days

		Size (mm)	Weight (mg)
	Z. annulosus
	First instar nymphs	4	2.8
	Second instar nymphs	5-6	6 . 0
	Third instar nymphs	8-10	17.0
	Fourth instar nymphs	12-14	35.5
	Fifth instar nymphs	18-20	63.0
	Adults	21-24	67.5
	Ratio between the weight of experimental prey and Z. annulosus nymphs
	Nasutitermes nigriceps workers	4-5 mm	9-12 mg
	First instar nymphs	3.21 to 4.28
	Second instar nymphs	1.50 to 2.00
	Third instar nymphs	0.53 to 0.71
	Fifth instar nymphs	0.14 to 0.19
	Tipulidae	27-30 mm	17-25 mg
	Third instar nymphs	1 to 1.47
	Grasshoppers	10-12 mm	35-44 mg
	Third instar nymphs	2.06 to 2.59
	Katydids	12-14 mm	80-100 mg
	Second instar nymphs	13.33 to 16.67
	Fourth instar nymphs	2.53 to 2.82
	Fifth instar nymphs	1.27 to 1.59
	Katydids	19-20 mm	135-150 mg
	Second instar nymphs	22.50 to 25.00
	Third instar nymphs	7.94 to 8.82

Table 2

 2 Different arthropod taxa captured under natural conditions by Zelus annulosus (N =308)

	a Attracted by a prey already in
	the process of being eaten by
	Zelus annulosus nymphs and
	captured in turn

Acknowledgments We are grateful to Andrea Yockey-Dejean for proofreading the manuscript and the Laboratoire Environnement de Petit Saut for furnishing logistical help. Financial support for this study was partially provided by a fellowship from the French Investissement d'Avenir grant managed by the Agence Nationale de la Recherche (CEBA, ref. ANR-10-LABX-0025), project Tri-Nutri, and by the Programme Convergence 2007-2013 Région Guyane (project Bi-APPLI) from the European Community.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

The experiments comply with the current laws of the country in which they were conducted.