F Lacam

J.-M Medjdoub

Routoure

Impact of the in situ SiN Thickness on Low-Frequency Noise in MOVPE

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Abstract-This paper reports on sub-10 nm quaternary barrier InAlGaN/GaN High Electron Mobility Transistors (HEMTs) grown by Metal-Organic-Vapor-Phase-Epitaxy (MOVPE) with an in situ SiN passivation layer, and ultra-short gate length of 200 nm. Two batches of HEMTs with two SiN thicknesses (t SiN ) of 14 and 22 nm are studied. Low Frequency Noise (LFN) measurements of the drain current have been carried out in the linear regime and showed that the in situ SiN thickness has no impact on the noise performance. S ID /I D 2 in the linear regime dependence over the gate overdrive shows that the channel noise is located under the gate and that the noise is not impacted by the thickness of the in situ SiN layer.

Index Terms-InAlGaN/GaN, in situ SiN passivation, low frequency noise, HEMT

I. INTRODUCTION

LTRA-THIN quaternary barrier InAlGaN/GaN High Electron Mobility Transistors (HEMTs) hold a tremendous potential for microwave and millimeter-wave applications, thanks to their outstanding 2-DEG properties that allowed to achieve state of the art electron mobility of 1800 cm 2 •V -1 •s -1 and sheet carrier density of 1.9•10 13 cm -2 at room temperature [START_REF] Medjdoub | High electron mobility in high polarization sub-10 nm barrier thickness InAlGaN/GaN heterostructure[END_REF]. This is owing to the use of Al-rich InAlGaN layers having an important effect to enhance the carrier density and achieve high frequency performance due to the increase of the spontaneous polarization [START_REF] Ketteniss | Study on quaternary AlInGaN/GaN HFETs grown on sapphire substrates[END_REF]- [START_REF] Aubry | ICP-CVD SiN Passivation for High-Power RF InAlGaN/GaN/SiC HEMT[END_REF].

One of the critical parameters limiting dynamic and power performance as well as the electrical reliability of GaN based HEMTs is the gate leakage current that might increase under high electric field [START_REF] Meneghesso | Reliability of GaN High-Electron-Mobility Transistors: State of the Art and Perspectives[END_REF]- [START_REF] Marcon | Reliability of AlGaN/GaN HEMTs: Permanent leakage current increase and output current drop[END_REF]. The parasitic effects leading to current collapse and gate leakage current increase have been reduced by using deposited SiN passivation [START_REF] Green | The Effect of Surface Passivation on the Microwave Characteristics of Undoped AlGaN/GaN HEMT's[END_REF]- [START_REF] Arulkumaran | Surface passivation effects on AlGaN/GaN high-electron-mobility transistors with SiO2, Si3N4 , and silicon oxynitride[END_REF]. This work was supported by National Research Agency under contract N° ANR-14-CE26-0022 (LHOM).

M. Rzin was with Normandie Univ., UNICAEN, ENSICAEN, CNRS, GREYC, 14000 Caen, France. He is now with DEI, University of Padova, Italy (e-mail: mehdi.rzin@ensicaen.fr).

B. Guillet, L. Méchin and J.-M. Routoure are with Normandie Univ., UNICAEN, ENSICAEN, CNRS, GREYC, 14000 Caen, France (e-mail: bruno.guillet@unicaen.fr; laurence.mechin@ensicaen.fr, jeanmarc.routoure@unicaen.fr).

P. Gamarra and C. Lacam are with III-V Lab, Thales Research and Technology, 91767 Palaiseau, France (e-mail: piero.gamarra@3-5lab.fr, cedric.lacam@3-5lab.fr).

F. Medjdoub is with IEMN, UMR8520, 59650 Villeneuve d'Ascq, France (e-mail: farid.medjdoub@iemn.univ-lille1.fr).

However, the leakage current remains a serious issue when ultra-thin barrier layers are used. In order to reduce the leakage current under the gate and/or in the gate-drain access region, the deposition of an in situ SiN passivation layer on top of the quaternary barrier in the Metal-Organic-Vapor-Phase-Epitaxy (MOVPE) reactor has been proposed and successfully applied.

The passivation layer deposited in the same growth run as the III-N heterostructure reduces the relaxation, cracking and surface roughness of the barrier layer [START_REF] Derluyn | Improvement of AlGaN/GaN high electron mobility transistor structures by in situ deposition of a Si3N4 surface layer[END_REF]. Moreover, the low growth rate and the relative high growth temperature of in situ SiN layer reduce the formation of surface states and improve the performance of the 2DEG properties.

The thickness of in situ SiN dielectric gate has a significant impact on the leakage current and may affect the drain current. Thick SiN cap increases the distance between the channel and the surface, thus reducing the surface state effects. On the other hand, much larger SiN cap makes the definition of ultra-short gate footprints more challenging.

Low frequency noise (LFN) measurements can be used to investigate the material/device quality, to identify material defects and trapping effects, and to investigate the reliability of GaN based HEMTs [START_REF] Mutta | Volume charge carrier number fluctuations probed by low frequency noise measurements in InN layers[END_REF]- [START_REF] Seo | A novel analysis of Lgd dependent-1/f noise in In0.08Al0.92N/GaN fin-HEMTs[END_REF]. In addition to significant reduction of the gate leakage current and the increase of the sheet charge density, the in situ SiN passivation layer can also potentially improve the low frequency device noise performance [START_REF] Vertiatchikh | Effect of the surface and barrier defects on the AlGaN/GaN HEMT low-frequency noise performance[END_REF]- [START_REF] Moens | An Industrial Process for 650V rated GaN-on-Si Power Devices using in-situ SiN as a Gate Dielectric[END_REF].

In this letter, we report on low frequency noise measurements performed on InAlGaN/GaN HEMTs with different MOVPE in situ SiN passivation thicknesses.

II. EXPERIMENTAL DETAILS

The SiN/InAlGaN/GaN HEMT structures were grown in an Aixtron Close Coupled Showerhead (CCS) MOVPE reactor on 4H-SiC semi-insulating substrates.

The growth details were published in Ref. [START_REF] Gamarra | M-A di Forte-Poisson "In-situ passivation of quaternary barrier InAlGaN/GaN HEMTs[END_REF]. First SiC substrate surfaces were prepared in H 2 and the growth was initiated with an AlN nucleation layer (100nm thick) at high temperature. A 1.6 μm thick highly resistive carbon doped GaN buffer layer, a 150nm thick non-intentionally doped GaN channel, an AlN interlayer with a nominal thickness of 1.

III. RESULTS AND DISCUSSION

Typical I D -V GS and g m -V GS characteristics at V DS = 200 mV in the linear regime of two representative InAlGaN/GaN HEMTs (A) with t SiN = 14 nm and L GD =1, 2 and 3 µm are shown in Fig. 2. The threshold voltage is around -1.6 V. These devices have an electron mobility of 1792 cm 2 •V -1 •s -1 and sheet carrier density of 1.64•10 13 cm -2 , resulting in a sheet resistance of 212 Ω/□. Fig. 3 shows I D -V GS and g m -V GS characteristics of two representative InAlGaN/GaN HEMTs (B) with t SiN = 22 nm and L GD = 2 and 3 µm. The threshold voltage is around -2.2 V. These devices have an electron mobility of 1772 cm 2 •V -1 •s -1 and sheet carrier density of 1.60•10 13 cm -2 , resulting in a sheet resistance of 220 Ω/□.

The gate and drain subthreshold leakage currents are between 1 and 10 µA/mm at V GS = -5V and V DS = 10 V, typical of the Schottky contact on GaN based HEMTs. The main difference between A and B HEMTs is the negative shift of the threshold voltage when increasing the SiN thickness from 14to 22 nm.

The power spectral density (PSD) of the drain current S ID was measured in the linear regime at V DS = 200 mV to identify the dominant noise sources in the channel with these two in situ SiN passivation thicknesses. Noise measurements were carried out on devices under stationary conditions and were reproducible. At low frequency, S ID varies as 1/f type spectra (in the 1 Hz to 1 kHz frequency range) and no generationrecombination noise components were observed. There is no significant impact of SiN thickness on the noise performance. S ID /I D 2 decreases with the gate overdrive as (V GS -V TH ) -3 . Similar S ID /I D 2 dependence over V GS -V TH indicating the dominant channel noise is located under the gate, as reported in previous works on GaAs MODFETs [START_REF] Peransin | 1/f noise in MODFET's at low drain bias[END_REF] and AlGaN/GaN HEMTs [START_REF] Rao | Simultaneous low-frequency noise characterization of gate and drain currents in AlGaN/GaN high electron mobility transistors[END_REF]. In agreement with these results, it can be pointed out that the current collapse level within these devices as assessed by pulsed measurements has been found to be similar regardless of the SiN cap layer thickness. We previously analyzed S ID /I D 2 dependence over the gatedrain spacing of InAlGaN/GaN MIS-HEMTs by considering two noise sources in the channel: channel noise under the gate and channel noise in the gate-drain access regions [START_REF] Rzin | Impact of gate-drain spacing on low frequency noise performance of in-situ SiN passivated InAlGaN/GaN MIS-HEMTs[END_REF].

Here, the source and drain contact resistances and the gatesource access region resistance are negligible, hence the noise sources of these resistances are not considered in the model. Following the procedure of [START_REF] Rzin | Impact of gate-drain spacing on low frequency noise performance of in-situ SiN passivated InAlGaN/GaN MIS-HEMTs[END_REF], the experimental data representing the relative drain current channel noise under the gate (S IRCH /I D 2 ) versus gate overdrive voltage (V GS -V TH ), in the linear regime (V DS = 200mV), of InAlGaN/GaN HEMTs with L GD = 1, 2 and 3 µm are shown in Fig. 5. The relative drain current channel noise under the gate S IRCH /I D 2 decreases with the gate overdrive as (V GS -V TH ) -3 with no significant impact of L GD . Similar dependence over V GS -V TH indicating the dominant channel noise is located under the gate, as reported in previous works [START_REF] Peransin | 1/f noise in MODFET's at low drain bias[END_REF][START_REF] Rao | Simultaneous low-frequency noise characterization of gate and drain currents in AlGaN/GaN high electron mobility transistors[END_REF]. In [START_REF] Rzin | Impact of gate-drain spacing on low frequency noise performance of in-situ SiN passivated InAlGaN/GaN MIS-HEMTs[END_REF], the channel noise under the gate was also the main contribution of the total channel noise for for low gate-drain access region spacing L GD < 10 µm.

  2 nm and a 6 nm thick InAlGaN barrier layer were subsequently deposited. It was checked by STEM-EDS (Scanning Transmission Electron Microscopy -Energy-Dispersive Spectrometry) and μ-Auger analyses that the Impact of the in situ SiN thickness on low frequency noise in MOVPE InAlGaN/GaN HEMTs M. Rzin, B. Guillet, L. Méchin, P. Gamarra, C. Lacam, F. Medjdoub and J-M. Routoure U quaternary barrier showed Ga content of about 8-10% and In content ranging from 10% to 12%. At last, the SiN passivation layer (14 nm or 22 nm, noted t SiN ) was deposited on top of these heterostructures without growth interruption. Scanning Probe Microscopy (SPM) in conductive mode shows that the targeted thicknesses for the passivation layers are sufficiently thick to act as insulating layers. Device processing was performed as follows: ohmic contacts were formed directly on top of the InAlGaN barrier layer by etching the in situ Si x N y layer using a Fluorine-based plasma. A Ti/Al/Ni/Au metal stack was used, followed by rapid thermal annealing at 875 °C. Device isolation was achieved by nitrogen implantation. Ohmic contact resistances extracted from linear transmission line model (TLM) structures were 0.3 Ω.mm on average. A Ni/Au T-gate of 200 nm (L G ) was defined by e-beam lithography. Finally, a 200 nm thick SiN layer has been deposited as passivation.

Fig. 1 .

 1 Fig. 1. (a) Schematic cross-section view and (b) optical microscope image of SixNy/InAlGaN/AlN/GaN HEMT with 2 finger gates. In this work, the studied devices have a Ni/Au T-gate of 200 nm length (L G ) with two finger gates of width W G = 25µm. and 3 different gate drain region spacings L GD (1, 2 and 3 µm). The schematic cross-section view and an optical microscope image of the HEMT heterostructure are displayed in figure 1.

Fig. 2 .

 2 Fig. 2. ID-VGS and gm-VGS of InAlGaN/GaN MIS HEMTs with tSiN = 14 nm and LGD = 1, 2 and 3 µm at VDS = 200 mV.

Fig. 3 .

 3 Fig. 3. ID-VGS and gm-VGS of InAlGaN/GaN MIS HEMTs with tSiN = 22 nm and LGD = 2 and 3 µm at VDS = 200 mV. The experimental points representing S ID /I D 2 versus gate overdrive voltage (V GS -V TH ), in the linear regime (V DS = 200 mV), of InAlGaN/GaN HEMTs with L GD =1, 2 and 3 µm are shown in Fig.4. There is no significant impact of SiN thickness on the noise performance. S ID /I D 2decreases with the gate overdrive as (V GS -V TH ) -3 . Similar S ID /I D 2 dependence over V GS -V TH indicating the dominant channel noise is located under the gate, as reported in previous works on GaAs MODFETs[START_REF] Peransin | 1/f noise in MODFET's at low drain bias[END_REF] and AlGaN/GaN HEMTs[START_REF] Rao | Simultaneous low-frequency noise characterization of gate and drain currents in AlGaN/GaN high electron mobility transistors[END_REF]. In agreement with these results, it can be pointed out that the current collapse level within these devices as assessed by pulsed measurements has been found to be similar regardless of the SiN cap layer thickness.

Fig. 4 .

 4 Fig. 4. Dependence of the relative PSD of the drain current SID/ID 2 on the gate overdrive (VGS-VTH) at f = 1 Hz, VDS = 200 mV and T = 300 K.

Fig. 5 .

 5 Fig. 5. Dependence of the relative drain current channel noise under the gate SIRCH/ID 2 on the gate overdrive (VGS-VTH) at f = 1 Hz, VDS = 200 mV and T = 300 K.


Romain R Garnier
email: romaing@princeton.edu

Sylvain S Gandon

Yannick Y Chaval

Nathalie N Charbonnel

Thierry T Boulinier

Evidence of cross-transfer of maternal antibodies through allosuckling in a mammal: potential importance for behavioral ecology

Keywords:

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

a b s t r a c t

The transfer of maternal antibodies is a critical mechanism for the early life survival of vertebrate newborns. In mammals, passive transfer of immune compounds can occur prenatally through the placenta and postnatally through the consumption of colostrum and milk. In social mammals, it has been hypothesized that allosuckling may be a way for pups to broaden and strengthen their passive access to antibodies after birth, but empirical evidence for this mechanism is still lacking. In order to investigate the potential for the occurrence of a cross-transfer of antibodies between pups exposed to several females, we bred in a common environment groups of two females Mongolian gerbils (Meriones unguiculatus), each previously injected with a different vaccine. Here we report the dynamics of passively acquired specific antibodies in the serum of newborns, showing that pups acquired antibodies from both females of a group. Our result provides the first experimental evidence of a cross-transfer between litters of passively acquired antibodies. We discuss how such evidence opens perspectives for exploring the potential importance of horizontal transfer of immunity in natural host-parasite systems and how this could be used as a tool to answer important behavioral ecology questions.

© 2012 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved.

Environmental conditions encountered by a newborn during early life are important for its fitness later in life [START_REF] Lindström | Early development and fitness in birds and mammals[END_REF]. Parasites, in particular, represent a major pressure on the population dynamics and evolution of their host species [START_REF] Grenfell | Ecology of Infectious Diseases in Natural Populations[END_REF][START_REF] Tompkins | Parasites and host population dynamics[END_REF]. The effect of parasites may potentially be even stronger in young vertebrates whose immune response is not completely functional at birth [START_REF] Frank | Immunology and Evolution of Infectious Disease[END_REF]. In vertebrates, mothers have the ability to provide the newborn with a protection critical for early life survival [START_REF] Boulinier | Maternal transfer of antibodies: raising immunoecology issues[END_REF][START_REF] Grindstaff | Immune function across generations: integrating mechanism and evolutionary process in maternal antibody transmission[END_REF]) by transferring some specific molecules produced as part of the acquired immune response, the antibodies (also termed immunoglobulins [Ig]; [START_REF] Brambell | The Transmission of Passive Immunity from Mother to Young[END_REF]. In mammals, this transfer occurs through the placenta before birth and/or via the colostrum and the milk after birth [START_REF] Baintner | Transmission of antibodies from mother to young: evolutionary strategies in a proteolytic environment[END_REF]. The amount of Igs transferred through the placenta is determined by the anatomical structure of the placenta [START_REF] Chucri | A review of immune transfer by the placenta[END_REF], and in particular by variations in the number of tissue layers between maternal and fetal bloods. Differences in Ig subtypes are also important to consider as only immunoglobulin G (IgG) can reach the newborns bloodstream [START_REF] Pastoret | Handbook of Vertebrate Immunology[END_REF]. Similarly, postnatal absorption of antibodies by newborns also reveals variations across mammalian species. For instance, in artiodactyls, no placental transfer of Igs is possible and colostrum absorption is critical for early life survival [START_REF] Halliday | Variation in immunoglobulin transfer from ewes to lambs[END_REF]. In contrast, placental transfer seems to be the dominant route for Igs transfer in primates while Igs in the colostrum and the milk may mostly play a role in the local protection of the gut [START_REF] Sadeharju | Maternal antibodies in breast milk protect the child from enterovirus infections[END_REF]. In rodents, both prenatal and postnatal absorption of antibodies appear to be important factors to ensure the survival of the newborn [START_REF] Gustafsson | Pregnancy in B-Celldeficient mice -postpartum transfer of immunoglobulins prevents neonatal runting and death[END_REF].

How newborns gain access to colostrum and milk may thus be important in terms of passive transfer of immunity in many mammalian species. An interesting way for newborns to get milk is through allosuckling, when young individuals feed from a different female than their biological mother. Allonursing is indeed widely reported in mammals [START_REF] Packer | A comparative analysis of non-offspring nursing[END_REF][START_REF] Roulin | Why do lactating females nurse alien offspring? A review of hypotheses and empirical evidence[END_REF]) and offspring could gain important immunological benefits by acquiring antibodies from various lactating females (immunological function of allosuckling hypothesis; [START_REF] Roulin | The immunological function of allosuckling[END_REF]. The potential for an immunological function of allosuckling has been suggested through the efficient transfer of antibodies from a foster mother to her non-biological offspring (e.g. [START_REF] Gustafsson | Pregnancy in B-Celldeficient mice -postpartum transfer of immunoglobulins prevents neonatal runting and death[END_REF][START_REF] Halliday | Prenatal and postnatal transmission of passive immunity to young rats[END_REF]). However, adoption in the laboratory does not reflect conditions encountered normally in social groups. In the wild, mothers and newborns are exposed to biological and foster mothers/pups at the same time. Mothers may thus choose which pups to suckle and pups may in turn choose from which females to get milk.

In order to investigate the potential for the occurrence of a cross-transfer of antibodies, we focused on a social mammal, the Mongolian gerbil (Meriones unguiculatus). In the wild, this species usually forms groups of one breeding pair associated with a number of subordinates. In the lab however, they can breed successfully in groups, with several females giving birth together [START_REF] French | Alloparents in the Mongolian gerbil -impact on long-term reproductive performance of breeders and opportunities for independent reproduction[END_REF]. Two groups of 12 "specific pathogen free" females (obtained from Janvier, Le Genet-St-Isle, France) were vaccinated twice at 8 and 12 weeks of age respectively against influenza (0.1 mL, intramuscular injection; Gripovac, Merial, France) or Borrelia burgdorferi (0.1 mL, subcutaneous injection; Merilym, Merial, France). Antibodies specific of Borrelia (females vaccinated against influenza) or specific of influenza (females vaccinated against Borrelia) remained at undetectable levels throughout the study.

After the booster vaccination, each female was housed during 2 weeks with a male. To prevent direct initial contact and limit aggression, we split the cages with transparent Plexiglas walls (drilled with holes of approximately 1 cm). We then switched individuals between each side of the separation everyday during a week to get them used to each other. At the end of the week, the separation was opened and contact between the male and the female was rendered possible. When aggressive interactions among adults were nevertheless recorded, individuals were immediately separated. The same procedure was repeated to match females by pairs based on their vaccination status (one female vaccinated against influenza and the other against Borrelia). The females were subsequently housed together without the males. Pups were thus born in an environment mimicking a natural social system and expected to gain access to the milk of both females and to display detectable levels for both antigens.

At birth, newborns were marked subcutaneously with an individual combination of tattoo ink dots. At 3 weeks of age, before the first marking faded, an additional numbered ear tag (Monel 1005-1, National Band & Tag Co., Newport, KY, USA) was used to ensure a long lasting individual marking of individuals. The same ear tags were also used for the marking of females throughout the experiment. To assess passive antibody acquisition, blood was regularly obtained from the newborns during the rearing period. At day 1 (i.e., within 24 h after birth, as cages were checked every day) and day 8, a blood sample (20-30 L) was obtained by cardiac puncture using an insulin syringe with 30 G needle. After eyes had opened, blood sampling consisted of a puncture each week (starting day 19, and up to day 48) in the retro-orbital venal sinus with a heparinized capillary tube (75 L), alternatively from the left and right eye. Blood was then stored in dry tubes and centrifugated within an hour. The plasma was collected and kept frozen at -20 • C pending analyses. This sampling protocol was chosen to follow the newborns for a sufficiently long period after weaning (between 21 and 30 day; [START_REF] Norris | Mortality form birth to weaning in the Mongolian gerbil Meriones unguiculatus[END_REF].

Antibody levels in newborns were measured using specific commercial Enzyme Linked Immuno-Sorbent Assays (ELISA). An indirect ELISA kit was used for influenza (ID Screen Antibody Influenza A Competition, ID Vet, Montpellier, France). The percentage of inhibition (PI) relative to a negative control was used as a measure of antibody level. High PI values indicate high plasma concentrations of specific antibodies against influenza. A direct ELISA kit was used for Borrelia analyses (Borrelia IgG + VlsE ELISA, IBL International GMBH, Hamburg, Germany). As this kit is designed for humans, we replaced the secondary antibody by a peroxidase conjugated rabbit anti-gerbil IgG (Immunology Consultants Laboratory, Portland, OR, USA). Optical density (OD) was used as a measure of antibody levels in the newborns and high OD values reflected high serum levels of Borrelia specific antibodies. Analyses were run once for each sample due to the limited amount of serum available and were limited to the 4 pups that reached adulthood as complete dynamics over the rearing period were required to assess the dynamics of the passive transfer of antibodies.

The present study complies with the guidelines set in the Guide for the care and use of laboratory animals (National Research Council 2011) and the protocol has been approved by the Animal Care and Use Committee -Languedoc Roussillon (project number CEEA-LR-1003). In addition, because of the repeated sampling of newborns required by the protocol, we chose not to include any control group in order to minimize the number of animals used.

Despite preventive measures to limit agonistic interactions, females showed high levels of aggressiveness toward males. In addition, high rates of cannibalism of females on pups occurred rapidly after birth. The latter made it impossible to keep track of the exact number of births over the 12 female pairs set up. For only one pair of females, seven pups were found alive at day 1. Three of these newborns died during their first week, but the two females were able to raise the four remaining pups to adulthood. At birth, three of those four pups had high anti-Borrelia antibody levels while the remaining one had a much lower level (Fig. 1). As IgG against Borrelia can be transmitted through the placenta in rodents [START_REF] Morshed | Transfer of antibody against Borrelia duttonii from mother to young in Ddy mice[END_REF], this result suggests that those three pups were born to the Borrelia vaccinated mother. The female vaccinated against influenza likely gave birth to the remaining one. Anti-Borrelia antibody levels showed a steep increase during the first week of age which can be explained by the acquisition of maternal antibodies from the milk. All individuals reached very high levels by 8 days of age. The antibody levels remained relatively stable between 8 and 19 days of age, which is expected as newborns fed on milk during this period. Afterwards, antibody levels decreased rapidly and reached non-detectable levels by 40 days of age. The dynamics was similar for anti-influenza antibodies throughout the rearing period of newborns (Fig. 2). However, at birth, all individuals had no detectable antibodies indicating that antibodies against the nucleoprotein of influenza viruses may not be transmitted through the placenta.

All newborns displayed at some point during the rearing period detectable antibody levels specifically directed against both Borrelia and influenza. Although there was no control group of offspring raised by unvaccinated mothers, the decay of antibody levels following weaning is not consistent with a stimulation of the immune system of the newborns. It rather fits the dynamics expected if the newborns passively acquired antibodies from both females. This result demonstrates the efficiency of allosuckling as a source of antibodies in newborns and provides evidence for a potential immunological function of allosuckling. The transmission of antibodies from a mother to foster offspring has been described before but this is the first time that cross-transfer of antibodies has been reported in a social context, when females can choose which young to suckle and when newborns can choose from which female to suck.

A number of challenges related to the breeding of gerbils have reduced the sample size in the present study. A way to reduce aggressiveness problems could be to rear non-sexually mature individuals in groups of one male and two females. Because the social group would not have been modified recently before the females give birth, lower levels of infanticide could be expected (e.g. [START_REF] Elwood | Ethical implications of studies on infanticide and maternal aggression in rodents[END_REF]. Another possibility is to pair females only after the pups were born. In this latter case, the identity of the mother of the pups would be known and one could thus avoid sampling newborns in the very few days following birth. However, such a design would limit the access of pups to the colostrum of non-biological mothers and in turn may modify the dynamics of acquisition of passive antibodies by the newborns.

The rapid decay of antibody levels starting after 20 days of age is associated with weaning, which usually occurs between 21 and 30 days in Mongolian gerbils [START_REF] Norris | Mortality form birth to weaning in the Mongolian gerbil Meriones unguiculatus[END_REF]. At that time, newborns stop feeding on milk and do not acquire passive immunity from their mothers. The transfer of antibodies from the intestinal lumen to the bloodstream of the newborn indeed relies on a receptor, the neonatal Fc receptor (FcRn) which has in mice been shown to be expressed in the gut only during the neonatal period [START_REF] Roopenian | FcRn: the neonatal Fc receptor comes of age[END_REF]. If and how this receptor is expressed in the intestine of newborn gerbils is unknown but it probably influences the acquisition of passive antibodies.

Our result suggests that newborns may receive antibodies from several lactating females of a social group. However, how newborns will have access to antibodies is likely to vary with the synchrony of births within groups. Offspring from the first litter, usually born to the dominant female, may for instance be able to broaden and strengthen their repertoire of antibodies with the colostrum of foster mothers. Conversely, pups born later to subordinate females would only gain access to a limited amount of antibodies from the dominant female's milk. The existence of an efficient cross-transfer of antibodies may also contribute to the decision of dominant females to allow subordinate breeding. In some social mammals, such as banded mongooses (Mungos mungo), subordinate females can also be forced out of the group during the course of the gestation [START_REF] Cant | Reproductive control via eviction (but not the threat of eviction) in banded mongooses[END_REF][START_REF] Gilchrist | Female eviction, abortion, and infanticide in banded mongooses (Mungos mungo): implications for social control of reproduction and synchronized parturition[END_REF]. Excluded females can subsequently be reintegrated in the group after aborting. Should this abortion occur late in gestation, it has the potential to result in spontaneous lactation which may further reduce the amount of maternal care required from the dominant female. In addition, subordinate females may experience a rather different parasitic environment during the course of exclusion and broaden the repertoire of the pups of the dominant female through cross-transfer of antibodies.

The use of vaccines [START_REF] Staszewski | Vaccination: a way to address questions in behavioral and population ecology?[END_REF]) as individual markers of females could allow the exploration of the cross-transfer of antibodies in the wild. If each female from a social group receives a different vaccine, antibodies against each vaccine could be searched for in newborns and used to assess the transfer of antibodies through allosuckling. The choice of the vaccines should be made carefully not only to ensure safety for the vaccinated individuals but also the availability of accurate ELISA techniques to measure specific antibody levels in the pups. A single blood sample of the newborn at a carefully chosen time would be sufficient to obtain evidence for the cross-transfer of antibodies. Finer resolution antibody dynamics could allow for the exploration of the effect of birth synchrony within groups. Social mammals such as meerkats [START_REF] Clutton-Brock | Cooperation, control, and concession in meerkat groups[END_REF], banded mongooses [START_REF] Cant | Social control of reproduction in banded mongooses[END_REF] or rodents (e.g. the Alpine marmot, Cohas and Allainé 2009) for which long-term monitoring programs are already implemented might prove particularly suitable for such studies.

Fig. 1 .

 1 Fig. 1. Borrelia specific antibody levels in the serum of the pups at different age. Antibody levels are expressed as optical densities (OD) measured by ELISA: a high value of OD corresponds to high antibody levels. Colors refer to the vaccination of the biological mother (grey for Borrelia, black for influenza) and symbols are specific of the individual. Plain curves represent the dynamics of antibodies acquired from the biological mother while dashed curves represent the dynamics of antibodies acquired through allosuckling.

Fig. 2 .

 2 Fig. 2. Influenza specific antibody levels in the serum of the pups at different age. Antibody levels are expressed as percentage of inhibition (PI) by comparison with a control sample: a high value of PI corresponds to high antibody levels. Colors refer to the vaccination of the biological mother (grey for Borrelia, black for influenza) and symbols are specific of the individual. Plain curves represent the dynamics of antibodies acquired from the biological mother while dashed curves represent the dynamics of antibodies acquired through allosuckling.

 No. of Pages 4

R. Garnier et al. / Mammalian Biology xxx (2012) xxx-xxx

Acknowledgements

The authors thank Audrey Arnal, Carine Brouat, Philippe Perret, Aurore Ponchon and Anne Xuereb for discussions and help at various stages of the experiment and two anonymous reviewers for comments on an earlier version of the manuscript. Technical help for the immunological analyses was provided by the PACE. The members of the "Terrain d'Expérience" platform at the CEFE-CNRS played an important role by designing and producing the Plexiglas separations. Financial support for this study came from the Centre National de la Recherche Scientifique (CNRS) and the Agence Nationale de la Recherche (ANR) grants 07 JCJC 0128 "EPICE" to SG and 11 BSV7 003 "EVEMATA" to TB. RG was supported by a PhD fellowship from the French Ministry of Research.


Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012 Comment citer ce document : Taillandier, P., Duchêne, C., Drogoul, A. (2011). Automatic revision of the control knowledge used by trial and error methods: Application to cartographic generalisation. Applied Soft Computing, 11 (2), 2818-2832. , DOI : 10.1016/j.asoc.2010.11.012


A. a , Capowiez, Y. b , Guérold, F. a , Nahmani J Auclerc
email: auclerca@umich.edu

Ac

Application of X-ray tomography to evaluate liming impact on earthworm burrowing activity in an acidic forest soil under laboratory conditions

Keywords:

1. Introduction

In the 1980s, important forest decline was observed in numerous sensitive regions throughout the Northern hemisphere due to acidic atmospheric depositions caused by human activities [START_REF] Campbell | Atmospheric deposition of sulphur and nitrogen species in the U[END_REF][START_REF] Driscoll | Acidic deposition in the Northeastern United States: sources and inputs, ecosystem effects, and management strategies[END_REF]. Acidic atmospheric deposition can decrease forest soil pH, indirectly induce earthworm abundance, and allow their replacement by enchytraeids in acidic soils [START_REF] Edwards | Biology and Ecology of Earthworms[END_REF][START_REF] Graefe | Critical values of soil acidification for annelid species and the decomposer community[END_REF][START_REF] Räty | Earthworm communities in birch stands with different origin in central Finland[END_REF]. To counteract increasing soil acidity and forest dieback, many European countries conducted large-scale liming [START_REF] Hüttl | Liming as a mitigation tool in Germany's declining forests-reviewing results from former and recent trials[END_REF][START_REF] Formanek | A contribution to the effect of liming on forest soils: review of literature[END_REF][START_REF] Godbold | Managing Acidification and Acidity in Forest Soils[END_REF]. Several studies showed an increase of earthworm populations following forest liming [START_REF] Kreutzer | Effects of forest liming on soil processes[END_REF][START_REF] Theenhaus | The effects of clear-cutting and liming on the soil macrofauna of a beech forest[END_REF][START_REF] Deleporte | Long-term effects of mineral amendments on soil fauna and humus in an acid beech forest floor[END_REF][START_REF] Potthoff | Earthworm communities in temperate beech wood forest soils affected by liming[END_REF]. Similarly, the introduction of Lumbricus terrestris, an epi-anecic species [START_REF] Judas | The inoculation of Lumbricus terrestris L. in an acidic spruce forest after liming and its influence on soil properties[END_REF]) and of the endogeic earthworm Aporrectodea caliginosa [START_REF] Robinson | Effects of earthworms on cation and phosphate mobilisation in limed peat soils under Picea sitchensis[END_REF] in limed acidic forest lead to the establishment of reproducing populations.

Despite the roles of earthworm species play in many soil processes [START_REF] Lavelle | Soil invertebrates and ecosystem services[END_REF], to our knowledge, few studies have addressed the impact of forest liming on earthworm activity [START_REF] Springett | Effect of pH and calcium content of soil on earthworm cast production in the laboratory[END_REF][START_REF] Robinson | Burrowing and soil consumption by earthworms in limed and unlimed soils from Picea Sitchensis plantations[END_REF][START_REF] Judas | The inoculation of Lumbricus terrestris L. in an acidic spruce forest after liming and its influence on soil properties[END_REF][START_REF] Auclerc | Adapting ecotoxicological tests based on earthworm behavior to assess the potential effectiveness of forest soil liming[END_REF]. By ingesting soil, fragmenting organic matter, mixing and ejecting casts above and below ground (i.e. casting and burrowing activities), earthworms increase nutrient cycling and microbial activity in organic and mineral soil horizons [START_REF] Jones | Organisms as ecosystem engineers[END_REF][START_REF] Shipitalo | Quantifying the effects of earthworms on soil aggregation and porosity[END_REF][START_REF] Lavelle | Soil invertebrates and ecosystem services[END_REF]. It is, however, difficult to study earthworm behaviour directly under field conditions because of the complex interactions between abiotic and biotic factors. Therefore, repacked soil is often used under laboratory conditions to describe burrowing behaviour of individual earthworm species, or to describe interspecific interactions between earthworm species [START_REF] Joschko | A non-destructive method for the morphological assessment of earthworm burrow systems in three dimensions by X-ray computed tomography[END_REF][START_REF] Jégou | Assessment of the burrow system of Lumbricus terrestris, Aporrectodea giardi, and Aporrectodea caliginosa using X-ray computed tomography[END_REF]3 Jégou et al., 2001;[START_REF] Capowiez | Burrow systems made by Aporrectodea nocturna and Allolobophora chlorotica in artificial cores: morphological differences and effects of interspecific interactions[END_REF][START_REF] Bastardie | X-ray tomographic and hydraulic characterization of burrowing by three earthworm species in repacked soil cores[END_REF]. X-ray tomography, as a nondestructive, accurate, and rapid method allows a global representation of the organisation of soil macroporosity [START_REF] Capowiez | Using X-ray tomography to quantify earthworm bioturbation non-destructively in repacked soil cores[END_REF]. In repacked soil cores, 3-D reconstructions of burrow systems using X-ray tomography have been used to assess changes in the earthworm burrowing behaviour under several conditions, including metal pollution [START_REF] Nahmani | Effects of metal pollution on soil macroinvertebrate burrow systems[END_REF], pesticide exposure [START_REF] Dittbrenner | Assessment of short and long-term effects of imidacloprid on the burrowing behaviour of two earthworm species (Aporrectodea caliginosa and Lumbricus terrestris) by using 2D and 3D post-exposure techniques[END_REF], or coal fly ash fertilisation [START_REF] Yunusa | Amendment of soil with coal fly ash modified the burrowing habit of two earthworm species[END_REF].

The present study aims to determine the impact of lime addition in an acidified forest soil on the burrowing behaviour of earthworm, by using X-ray tomography. Lime addition is an increasingly used management practice in the Vosges Mountains (North-eastern, France) to counteract soil acidity and increase forest soil fertility [START_REF] Landmann | Forest Decline and Atmospheric Deposition Effects in the French Mountains[END_REF][START_REF] Bonneau | Evolution of the mineral fertility of an acidic soil during a period of ten years in the Vosges Mountains (France). Impact of humus mineralisation[END_REF][START_REF] Van Der Heijden | Longterm sustainability of forest ecosystems on sandstone in the Vosges Mountains (France) facing atmospheric deposition and silvicultural change[END_REF]. The objectives of this study include: 1) quantification of earthworm burrow structural features (mean diameter, burrow number, burrow distribution by soil depth) of the endogeic Aporrectodea caliginosa Savigny and the anecic A. giardi Ribaucourt species in limed and non-limed soils and 2) comparison of the burrowing and surface casting activities of these earthworms in recent (in vitro) versus prior (in situ) limed soils. We expect an increase of earthworm activities (burrowing and casting) by in vitro and in situ lime addition into the acidic forest soil. To this extent, we used cores with repacked soil sampled in the Vosges Mountains under laboratory conditions.

Material and Methods

Soils and earthworms

4 Soils used in this study were collected from two granitic forest catchments dominated by beech (Fagus sylvatica) located in the Vosges Mountains (North-eastern, France): a nonlimed control catchment (47°57'39.5''N -006°53'05.1''E; Altitude: 1060 m), and an adjacent, limed catchment (47°57'22.9''N -006°52'55.6''E, Altitude: 1045 m). The calcareous amendment (dolomite) was spread by helicopter in October 2003 (i.e. 6 years before the laboratory study) as a fine powder composed of 70 % CaCO 3 , 17 % MgCO 3 , 10 % CaSO 4 and 3 % KCl at 2.5 t ha -1 .

Catchment soils are Entic Podzols according to WRB [START_REF] Wrb Classification | World Reference Base for Soil Resources[END_REF]. Soil texture in the upper 30 cm of the non-limed catchment was 55.8 % sand, 22.4 % silt, and 21.8 % clay. It contained 29.3 % organic matter and had WHC of 0.16 g g -1 . The mean pH (measured in water) of the OH horizon was 3.8, and the A horizon was 4.5. Soil texture in the upper 30 cm of the limed catchment was 58.7 % sand, 22.9 % silt, 18.4 % clay. It contained 23.3 % organic matter and had WHC of 0.18 g g -1 . Mean pH in this treated catchment was 4.1 in the OH horizon and 4.7 in the A horizon.

The OH and A horizons of non-limed and limed catchments were sampled. Soils for the nonlimed control came from the non-limed catchment, and soils for the in situ limed treatment came from the limed catchment. After field collections, OH and A horizons were sieved through 3.9 mm mesh and stored at 12°C for one week before repacking for the experiment.

The two earthworm species studied belong to two different ecological categories: A. giardi is anecic and A. caliginosa is endogeic. Adult earthworms were collected by handsorting from an experimental plot in La Bouzule (48°74'10.3''N -006°32'59''E). For acclimation, earthworms were stored at 12°C for one week with 200 g of beech litter and 5 kg of non-limed OH and A horizon soil prior the start of the experiment. 5

Experimental procedure

The experiment was performed using PVC cylinders (15.5 cm diameter, 30 cm long) with a 1 mm nylon mesh at the bottom to allow the passage of gas and water. Three soil treatments (i.e. non-limed, in situ and in vitro limed) were established by filling the PVC cylinders with 20 cm of sieved A horizon soil, to which 5 cm of sieved OH horizon soil was added (Table 1a). For the in vitro limed soil treatment, we used OH horizon soil collected from the non-limed catchment into which we mixed 5 g of lime (mean pH = 5.4), which is equivalent to the 2003 catchment-level application of 2.5 t ha -1 . We placed this freshly limed soil above sieved A horizon soil (mean pH = 4.5) from the non-limed catchment.

Field soil bulk density (0.8 g cm -3 ) was achieved by repacking the soil and pressing it for 5 minutes with a hydraulic press at 0.75 bar. Seven layers of 0.7 kg each were sequentially added to reproduce the A horizon. Two layers of 0.4 kg each were added and packed to reproduce the OH horizon. Soil cores were acclimated in a climate chamber at 12°C ± 1°C for two days before earthworm inoculation.

Each core of three replicates per treatment was inoculated with either two individuals of A. giardi or two individuals of A. caliginosa (Table 1b). In total, 36 earthworms were applied. Individual A. giardi earthworms initially weighed 2.56 ± 0.3 g (mean ± SD, fresh weight) and A. caliginosa individuals weighed 0.73 ± 0.1 g. We prepared one earthworm-free control core for each soil treatment. To each core, after inoculation with earthworms, we added 5 g (fresh mass) of beech leaves -sampled from the litter layer of the non-limed and limed catchments -as a food source. We also added 150 mL of distilled water in each core to remoisten the soil and the litter during the experiment. All cores were kept in a climate 6 chamber at 12°C ± 1°C with a light/dark cycle of 16/8h for 9 weeks from November 2009 to January 2010.

3-D reconstruction of the burrow system and topology analysis

After the 9 weeks incubation, each column was scanned at the INRA Nancy Centre using a medical X-ray tomograph (General Electrics; brightspeed exel) according to the method described in [START_REF] Capowiez | 3D skeleton reconstructions of natural earthworm burrow systems using CAT scan images of soil cores[END_REF]. Columns were scanned horizontally at 130kV and 80 mA s -1 , providing 2-D images (1.25 mm thick every 1.25 mm) of the soil macroporosity. After a simple binarization of the 2-D images presenting two well separated peaks in the grey level histogram, the 3-D volume reconstruction was run by a specific algorithm [START_REF] Pierret | 3D reconstruction and quantification of macropores using X-ray computed tomography and image analysis[END_REF]. The 3-D skeletons were also reconstructed by connecting the centroids of pores that overlapped between two successive images [START_REF] Capowiez | 3D skeleton reconstructions of natural earthworm burrow systems using CAT scan images of soil cores[END_REF].

Density parameters were analysed to obtain several characteristics: number of burrows, total burrow length, and total burrow volume. In order to evaluate the earthworm activity throughout the soil core, the cumulative burrow volume (cm 3 ) was summed to 25 cm depth.

We use the slope of cumulative volume by depth relationship (expressed in cm 3 cm -1 ) as a measure of earthworm activity.

Surface earthworm casts, soil core pH and earthworm biomass

After column scanning, earthworm cast production (CP, expressed as mg of dry casts g -1 of fresh earthworm initial weight d -1 ) was measured by collecting surface casts drying at 60°C for 3 days, and weighing. Afterward, each column was destructively harvested by layer and earthworms were hand-sorted. Earthworm survival was assessed by gently prodding dried with filter paper and weighed. Soil pH was measured by soil horizon at the end of the experiment using a 1:5 soil:water ratio.

Statistical analysis

Student t-tests or Welch tests (when the homogeneity of variance was not verified)

were used to assess effects of soil and earthworm treatments on each burrow system characteristic, cast production, slope of the cumulative burrow-depth relationship, and final pH. Paired t-tests assessed the difference between initial and final pH for each individual depth of soil and earthworm treatment. All analyses were conducted using R software (R Development Core Team, 2008).

Results

Soil core pH

At the beginning of the experiment (T0), after lime addition into the upper 5 cm of the in vitro limed soils (OH horizon), pH was 5.4 and was significantly greater than for the other soil treatments (P < 0.01; Table 2). The pH of the upper 5 cm soil of the in situ limed treatment was also significantly greater than in the non-limed treatment (P = 0.03; Table 2).

These differences remained at the end of experiment (Tf) for each earthworm treatment (P < 0.01) and in control incubations (Table 2). In the A layer at T0, the pH of the in situ limed treatment was significantly greater than in the two other treatments (P < 0.01). The final (Tf) pH in the A layer of the A. giardi treatment was similar to T0 (P = 0.19) and Tf values were similar across soil treatments (P = 0.10). For the A. caliginosa treatment, the final A layer pH in the in situ limed treatment was significantly smaller than at the start of incubation (P = 0.02), but pH increased in the in vitro limed treatment during the experiment (P = 0.04).

Final earthworm biomass and surface cast production

All earthworms were alive at the end of experiment, as indicated by physical reactions to Pasteur pipet stimulation. Biomass of both species decreased during the experiment for each soil treatment (Table 3). A. giardi lost significantly more weight in the non-limed treatment than in either limed treatment (P = 0.02 for non-limed vs. in situ limed and P = 0.01 for non-limed vs. in vitro limed treatment). They lost 59.2 % of initial biomass in the nonlimed soil and 13.7 to 23.2 % in the limed treatments. For A. caliginosa the mass loss was not significantly different between the soil treatments and ranged from 12.2 % to 22.3 % (Table 3).

Cast production (CP) by A. giardi in the in vitro limed treatment was significantly higher than in the non-limed treatment (P < 0.01) but not different from the in situ limed treatment (P = 0.30; Table 3). Surprisingly, the endogeic worm A. caliginosa seemed to produce more surface casts than A. giardi. Anyway, no significant differences were found for A. caliginosa cast production between soil treatments and its production ranged from 63.6 to 109.6 mg g -1 d -1 (Table 3).

Burrowing patterns

3-D burrow system characteristics

Burrow networks occupied the entire column depth for all earthworm treatment (Fig. 1). A. giardi built large-volume, vertically oriented burrows, while A. caliginosa burrow 9 volumes were smaller and more horizontal. A. giardi burrowed a significantly higher total volume in the in vitro limed treatment than in the non-limed treatment (P < 0.01; Table 4), whereas the volume burrowed in the in situ treatment was not significantly different from the non-limed treatment. The same pattern was observed for the total burrow length (P = 0.01).

No significant liming effect was found for the total burrow length or volume burrowed by A.

caliginosa (P > 0.05; Table 4). Moreover, A. giardi burrowed a significantly higher volume in the in vitro limed treatment than A. caliginosa (Table 4; P < 0.01). However, A. caliginosa excavated 2 to 3 times longer (m g -1 of initial earthworm biomass; P < 0.05) and 2 to 5 times more burrows (P < 0.04) than A. giardi. Finally, the mean burrow length dug in the OH horizon (top 5 cm) was higher in the in vitro limed treatment than in either other soils for both species (Table 4; Fig. 1).

Mean cumulative volume of burrows as a function of depth

The mean cumulative burrow volume versus depth relationship differed by soil treatment and species (Fig. 2). In the in situ limed and non-limed treatment, burrow volume increased linearly as a function of core depth regardless of species. In the in vitro limed treatment, this relationship showed two phases: first, a rapid and linear increase of burrow volume in the 0-5 cm layer (OH horizon), and second, a linear but slower excavated volume increase for 5-20 cm depth (A horizon). The relationship was significantly different between the in vitro limed and non-limed soil treatments for A. giardi in the OH horizon (P = 0.02),

with the mean slope of 0.28 for non-limed, 0.17 for in situ limed, and 0.06 cm 3 cm -1 for in vitro limed soils. For A. caliginosa, the same difference was denoted (P = 0.04; mean slope: 0.61; 0.33 and 0.17 cm 3 cm -1 in the non-limed, in situ and in vitro limed treatments, respectively). In the A horizon, no significant difference was found for either species across

Discussion

As expected, adding lime in the laboratory to acidified forest soils collected from the Vosges Mountains increased soil pH of the OH horizon from 4.0 to 5.4. During the 9 weeks of experiment, the lime mixed into the surface horizon affected deeper soil pH, especially in the presence of the endogeic earthworm A. caliginosa. [START_REF] Chan | Complementary ability of three European earthworms (Lumbricidae) to bury lime and increase pasture production in acidic soils of south-eastern Australia[END_REF] found that A.

caliginosa, A. longa, and A. trapezoides incorporated surface applied lime after 18 months in an acidic Australian pasture soil. Ingestion and deposition of casts below ground or by adherence on earthworm bodies transported the lime and incorporated it into deeper layers. In our laboratory experiment, we mixed the dolomite into the OH horizon to immediately assess its effect on the burrowing behaviour of earthworms. In the forest, dolomite is usually applied at the soil surface without any mixing. However, we expect that it leaches after dissolution and/or is incorporated by endogeic and anecic earthworm activities in deeper soil layers, inducing an increase of soil pH and of nutrient availability.

All earthworms of each species used in our experiment survived in the acidic soil for 9

weeks. As such, soil acidity at pH values as low as 3.8 were not lethal during this experiment. 11 should be less than 30 % during long-term experiments if we are to draw valid conclusions from the experimental results. For both studied species, the inoculated earthworms lost between 12 and 23 % except for the A. giardi which lost 59 % of body mass in the non-limed soil. Thus, without the lime, soil pH will likely limit A. giardi activity. [START_REF] Räty | Growth of Lumbricus terrestris and Aporrectodea caliginosa in an acid forest soil, and their effects on enchytraeid populations and soil properties[END_REF] described A. caliginosa as a eurytopic species inhabiting a variety of habitats. Sparse populations of this species have been found at pH 3.6 [START_REF] Bouché | Application of X-ray tomography to evaluate liming impact on earthworm burrowing activity in an acidic forest soil under laboratory conditions[END_REF]. This acid-tolerant species was also found in or introduced to and survived in soils with pH values from 3.9 to 7.0 [START_REF] Robinson | Effects of earthworms on cation and phosphate mobilisation in limed peat soils under Picea sitchensis[END_REF][START_REF] Chan | Complementary ability of three European earthworms (Lumbricidae) to bury lime and increase pasture production in acidic soils of south-eastern Australia[END_REF][START_REF] Räty | Growth of Lumbricus terrestris and Aporrectodea caliginosa in an acid forest soil, and their effects on enchytraeid populations and soil properties[END_REF][START_REF] Räty | Earthworms and pH affect communities of nematodes and enchytraeids in forest soil[END_REF][START_REF] Räty | Growth of Lumbricus terrestris and Aporrectodea caliginosa in an acid forest soil, and their effects on enchytraeid populations and soil properties[END_REF][START_REF] Potthoff | Earthworm communities in temperate beech wood forest soils affected by liming[END_REF]. A. giardi has been described as neutrophilic but tolerant to acidity and can live in soil with low organic matter [START_REF] Bouché | Application of X-ray tomography to evaluate liming impact on earthworm burrowing activity in an acidic forest soil under laboratory conditions[END_REF]. In a laboratory study, [START_REF] Salmon | Distribution of Heteromurus nitidus (Hexapoda, Collembola) according to soil acidity: interactions with earthworms and predator pressure[END_REF] successfully introduced this species in a forest eumoder, in an oligomull and in a calcic eumull with soil pH ranging from 4.0 to 7.4. In our study, A. giardi appeared sensitive to a small pH change and lime addition was likely to improve the habitat conditions since the species lost less biomass in limed soil. This biomass loss may also be explained by the low food quality of beech leaves (protein, carbohydrates, lignin content; [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF]. This concept is also highlighted by [START_REF] Vahder | Effect of pure and multi-species beech (Fagus sylvatica) stands on soil characteristics and earthworms in two northern German forests[END_REF] in two German forests where beech dominance was negatively correlated with the soil pH and endogeic earthworm biomass. The authors suggest that this pattern may be explained by the low quality and digestibility of beech litter.

The observed burrowing activity of A. caliginosa was typical for endogeic species:

narrow, horizontal and long burrows [START_REF] Jégou | Assessment of the burrow system of Lumbricus terrestris, Aporrectodea giardi, and Aporrectodea caliginosa using X-ray computed tomography[END_REF][START_REF] Jégou | Interactions between earthworm species in artificial soil cores assessed through the 3D reconstruction of the burrow systems[END_REF][START_REF] Langmaack | Quantitative analysis of earthworm burrow systems with respect to biological soil-structure regeneration after soil compaction[END_REF][START_REF] Bastardie | A new simulation for modelling the topology of earthworm burrow systems and their effects on macropore flow in experimental soils[END_REF][START_REF] Dittbrenner | Assessment of short and long-term effects of imidacloprid on the burrowing behaviour of two earthworm species (Aporrectodea caliginosa and Lumbricus terrestris) by using 2D and 3D post-exposure techniques[END_REF]. In the non-limed and in situ limed soils, this species did not show any depth preference as the burrows were found at every depths of the core. This contrasts with [START_REF] Jégou | Assessment of the burrow system of Lumbricus terrestris, Aporrectodea giardi, and Aporrectodea caliginosa using X-ray computed tomography[END_REF] who found A. caliginosa burrowed to a maximum 12 depth of 13 cm but most activity was in the top 7-8 cm in a laboratory incubation with repacked cultivated soil from Brittany, France kept for 246 days. [START_REF] Mckenzie | Size and orientation of burrows made by the earthworms Aporrectodea rosea and A. caliginosa[END_REF] observed that the most complex region of the burrow network of A. caliginosa was located between 2.5 and 5 cm. Others have found A. caliginosa active in both the topsoil and subsoil [START_REF] Joschko | A non-destructive method for the morphological assessment of earthworm burrow systems in three dimensions by X-ray computed tomography[END_REF][START_REF] Pitkänen | Distribution and abundance of burrows formed by Lumbricus terrestris L. and Aporrectodea caliginosa Sav. in the soil profile[END_REF][START_REF] Francis | The effects of three earthworm species on soil macroporosity and hydraulic conductivity[END_REF][START_REF] Langmaack | Quantitative analysis of earthworm burrow systems with respect to biological soil-structure regeneration after soil compaction[END_REF]. This shows that the burrowing behaviour of A. caliginosa is extremely dependent on soil characteristics and highlights the need for further earthworm burrow characterization. Furthermore, our study demonstrated that A. caliginosa had a depth preference for the topsoil (0-5 cm) after in vitro lime addition, which increased the pH in that depth from 4.0 to near 5.5.

In vitro liming stimulated A. giardi burrowing as evidenced by increased surface casting and increased soil excavation. These increases were most prominent in the top 5 cm.

The burrow system produced by the anecic A. giardi was characterised by approximately 20 burrows; and thus differed from the single, permanent, nearly vertical channels described for the well-studied epi-anecic Lumbricus terrestris, [START_REF] Jégou | Assessment of the burrow system of Lumbricus terrestris, Aporrectodea giardi, and Aporrectodea caliginosa using X-ray computed tomography[END_REF][START_REF] Shipitalo | Quantifying the effects of earthworms on soil aggregation and porosity[END_REF][START_REF] Bastardie | 3D characterisation of earthworm burrow systems in natural soil cores collected from a 12-year-old pasture[END_REF]. In their 246 day incubation of a cultivated soil from the deep mineral horizon, [START_REF] Jégou | Assessment of the burrow system of Lumbricus terrestris, Aporrectodea giardi, and Aporrectodea caliginosa using X-ray computed tomography[END_REF] found that the burrow system excavated by two individuals of A. giardi often reached the bottom of the column and maximal activity occurred between 4 and 23 cm. In our study, A. giardi did not show any depth preference in the nonlimed and in situ soil treatments, but it had increased activity and excavated longer burrows after in vitro liming, which induced the soil pH increase from 4.0 to near 5.5.

When comparing the burrow system of the two species regardless of soil pH, A.

caliginosa burrows were at least twice the length of burrows by A. giardi when standardized by biomass. This contrasts markedly with the 246 day laboratory incubation by [START_REF] Jégou | Interactions between earthworm species in artificial soil cores assessed through the 3D reconstruction of the burrow systems[END_REF], who found in a cultivated soil, that A. caliginosa burrows were four times shorter than 13 in our study, and that A. giardi burrowed approximately the same length as in our study, and thus more than A. caliginosa. This may be due to differing soil nature and soil bulk density:

1.35 g cm -3 in their study versus 0.8 g cm -3 in our study. Perhaps, the worms were also searching more for food or favorable conditions [START_REF] Jégou | Characterization of the burrow system of the earthworms Lumbricus terrestris and Aporrectodea giardi using X-ray computed tomography and image analysis[END_REF].

Finally, for both species, the volume, length, and number of burrows did not differ between in situ limed and non-limed soil. The burrow systems were similar even though A.

giardi lost less biomass and produced more casts in the in situ limed treatment than in the non-limed soil. Thus, six years after field liming, the management practice did not improve the burrowing activities of either of the earthworm species studied here. This may be explained by the remaining acidic conditions of the in situ limed soil, which is consistent with another laboratory study that investigated the impact of liming on the casting activity of Lumbricus terrestris in soils of the same stand as the present paper [START_REF] Auclerc | Adapting ecotoxicological tests based on earthworm behavior to assess the potential effectiveness of forest soil liming[END_REF]. Our previous work showed that the liming needed to increase soil pH to 5.3 under laboratory conditions in order to enhance earthworm activity, which is greater than the non-and in situlimed soils of the present study.

Conclusion

The use of X-ray computed tomography provides insights into understanding of the impacts of liming (a restoration method used in declined forests) on the burrowing activities of earthworm species which function as soil engineers. Liming affected the burrowing activity of the anecic A. giardi more than the endogeic A. caliginosa after a short term limed application (in vitro limed soil). In contrast, liming in the field six years prior to experiment Final earthworm biomass (% of initial earthworm fresh biomass) and final surface casts (mg g -1 day -1 ) by species and soil treatments. Masses are means (SD, n=3) with asterisk denoting significant difference (P<0.05) by species and letters denoting significantly (P<0.05) different soil treatments within earthworm species (t-or Welsh-tests).

  Comment citer ce document : Auclerc, A. . (Auteur de correspondance), Capowiez, Y., Guérold, F. .,Nahmani, J. (2013). Application of X-ray tomography to evaluate liming impact on earthworm burrowing activity in an acidic forest soil under laboratory conditions. Geoderma, 202, 45-50. DOI : 10.1016/j.geoderma.2013.03.011 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in: Geoderma (2012), Vol. 202, p. 45-50, DOI: 10.1016/j.geoderma.2013.03.011 Journal homepage: www.elsevier.com/locate/geoderma 7 individuals using a Pasteur pipet. Earthworms were then rinsed with distilled water, gently

  Comment citer ce document : Auclerc, A. . (Auteur de correspondance), Capowiez, Y., Guérold, F. .,Nahmani, J. (2013). Application of X-ray tomography to evaluate liming impact on earthworm burrowing activity in an acidic forest soil under laboratory conditions. Geoderma, 202, 45-50. DOI : 10.1016/j.geoderma.2013.03.011 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in: Geoderma (2012), Vol. 202, p. 45-50, DOI: 10.1016/j.geoderma.2013.03.011 Journal homepage: www.elsevier.com/locate/geoderma 10 treatments (0.43; 0.30; 0.42 and 0.53; 0.66; 0.68 cm 3 cm -1 in the non-limed, in situ and in vitro limed treatments for A. giardi, A. caliginosa, respectively). In the OH horizon, the slope was significantly smaller for A. giardi than A. caliginosa in the in vitro limed treatment (P < 0.01).

  Comment citer ce document : Auclerc, A. . (Auteur de correspondance), Capowiez, Y., Guérold, F. .,Nahmani, J. (2013). Application of X-ray tomography to evaluate liming impact on earthworm burrowing activity in an acidic forest soil under laboratory conditions. Geoderma, 202, 45-50. DOI : 10.1016/j.geoderma.2013.03.011 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in: Geoderma (2012), Vol. 202, p. 45-50, DOI: 10.1016/j.geoderma.2013.03.011 Journal homepage: www.elsevier.com/locate/geoderma

  Figure captions

Figure 2

 2 Figure 2Mean cumulative burrow volume by soil depth for (a) Aporrectodea giardi and (b) Aporrectodea caliginosa.

45-50, DOI: 10.1016/j.geoderma.2013.03.011 Journal homepage: www.elsevier.com/locate/geoderma 8

Comment citer ce document : Auclerc, A. . (Auteur de correspondance), Capowiez, Y., Guérold, F. .,

Nahmani, J. (2013)

. Application of X-ray tomography to evaluate liming impact on earthworm burrowing activity in an acidic forest soil under laboratory conditions. Geoderma, 202, 45-50. DOI : 10.1016/j.geoderma.2013.03.011 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in: Geoderma (2012), Vol. 202, p.

45-50, DOI: 10.1016/j.geoderma.2013.03.011 Journal homepage: www.elsevier.com/locate/geoderma

However, in the suboptimal experimental conditions, all individuals lost weight with A. giardi losing more weight than A. caliginosa. Experimental conditions recommended by

[START_REF] Fründ | Using earthworms as model organisms in the laboratory: Recommendations for experimental implementations[END_REF]

were carefully followed in our study. They suggest that control earthworm weight loss Comment citer ce document : Auclerc, A. . (Auteur de correspondance), Capowiez, Y., Guérold, F. ., Nahmani, J. (2013). Application of X-ray tomography to evaluate liming impact on earthworm burrowing activity in an acidic forest soil under laboratory conditions. Geoderma, 202, 45-50. DOI : 10.1016/j.geoderma.2013.03.011 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in: Geoderma (2012), Vol. 202, p.

2012), Vol. 202, p. 45-50, DOI: 10.1016/j.geoderma.2013.03.011 Journal homepage: www.elsevier.com/locate/geoderma 21Table 1

 1 Laboratory experiment soil source, lime addition, and earthworm treatments: (a) liming soil treatments by depth (b) total initial mass of two individual earthworms introduced to soil replicates by species.

	(a)
	Soil treatments	Soil in 0-5 cm	Soil in 5-25 cm
	non-limed	non-limed OH horizon	non-limed A horizon
	in situ limed	in situ limed OH horizon	in situ limed A horizon
	in vitro limed	non-limed OH horizon + 5 g lime	non-limed A horizon
	(b)
	Earthworm		Total biomass inoculated (Mean (SD)
	treatments	Species	initial fresh weight, g)
	Anecic	Aporrectodea giardi (2 individuals)	5.27 (0.61)
	Endogeic	Aporrectodea caliginosa (2 individuals) 1.52 (0.20)

Application of X-ray tomography to evaluate liming impact on earthworm burrowing activity in an acidic forest soil under laboratory conditions. Geoderma, 202, 45-50. DOI : 10.1016/j.geoderma.2013.03.011 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in: Geoderma (Comment citer ce document : Auclerc, A. . (Auteur de correspondance), Capowiez, Y., Guérold, F. ., Nahmani, J. (2013). Application of X-ray tomography to evaluate liming impact on earthworm burrowing activity in an acidic forest soil under laboratory conditions. Geoderma, 202, 45-50. DOI : 10.1016/j.geoderma.2013.03.011 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in: Geoderma (

2012), Vol. 202, p. 45-50, DOI: 10.1016/j.geoderma.2013.03.011 Journal homepage: www.elsevier.com/locate/geoderma 22Table 2

 2 Initial (T0) and final (Tf) soil pH by earthworm species treatment, soil liming treatment, and depth. Soil pH values are means (SD, n=3) with

	asterisks denoting significant differences between T0 and Tf (paired t-test) and letters denoting significant differences (P<0.05) for soil treatment
	nested within earthworm treatment*time (t-or Welsh-tests).
	Species		T0			Tf			Tf			Tf
	Treatments					Anecic A.giardi	Endogeic A.caliginosa		Control
	Soil	non-	in situ	in vitro	non-	in situ	in vitro	non-	in situ	in vitro	non-	in situ
	Treatments	limed	limed	limed	limed	limed	limed	limed	limed	limed	limed	limed

2012), Vol. 202, p. 45-50, DOI: 10.1016/j.geoderma.2013.03.011 Journal homepage: www.elsevier.com/locate/geoderma 24Table 4

 4 Comment citer ce document : Auclerc, A. . (Auteur de correspondance), Capowiez, Y., Guérold, F. .,Nahmani, J. (2013). Application of X-ray tomography to evaluate liming impact on earthworm burrowing activity in an acidic forest soil under laboratory conditions. Geoderma, 202, 45-50. DOI : 10.1016/j.geoderma.2013.03.011Version définitive du manuscrit publiée dans / Final version of the manuscript published in:Burrow system characteristics by earthworm species and soil liming treatments. Metrics are means (SD, n=3) with asterisks denoting significantly different (P<0.05) earthworm treatments and letters denoting significantly different soil liming treatments within earthworm species (t-or Welsh-tests).

	Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript	Species Treatment Species Treatment Soil Treatment Burrow Abundance Soil Treatment (#) Final earthworm biomass (% initial fresh biomass) Surface cast production (mg g -1 day -1 ) OH horizon Burrow Length (m) Total Burrow Length (m) Total Burrow Length per g Worm non-limed 40.8 (16.3) 76.8 (5.5) Tf Anecic A.giardi Tf Anecic A.giardi Non-limed in situ limed in vitro limed 12 (6.0) 21.7 (4.0) 26.7 (10.5) in situ limed in vitro limed non-limed * a * a a 86.3 (5.3) 84.3 (10.6) 77.7 (9.5) Endogeic A.caliginosa Tf Non-limed in situ limed in vitro limed Tf 62.3 (16.0) 51 (16.5) 66 (4.0) 31 32 Endogeic A.caliginosa in situ limed a a a in vitro limed 87.8 (9.7) *a b b a a a 28.6 (21.0) 97.5 (48.1) 136.7 (26.6) 63.6 (20.2) 109.7 (73.8) 87.4 (26.8) a ab b a a a 1.7 (0.7) 2.8 (0.5) 10.3 (6.4) 1.6 (0.9) 1.9 (0.9) 4.5 (0.4) a a a a a b 5.4 (2.0) 8.7 (5.3) 17.7 (4.3) 9.5 (1.4) 6.9 (3.3) 9.6 (2.1) * a ab * b a a a (m g -1 initial earthworm biomass) 1.0 (0.3) 1.6 (0.9) 3.4 (0.9) 6.6 (2.2) 4.3 (1.6) 6.5 (2.1) * a a b a a a Total Burrow Volume 54.6 (17.7) 75.3 (45.9) 116.3 (6.3) 46.3 (10.9) 36.8 (18.1) 52.9 (13.0) (cm 3 ) a ab * b a a a
	Manuscrit d'auteur / Author manuscript

Geoderma (

cm

Acknowledgments

This project was carried out with financial support from the National Research Agency (ANR) and Zone Atelier Moselle (ZAM). We thank Charline Freyburger from the National Institute of Agronomic Research (INRA) of Nancy, for her help with the tomography study and the technical staff of INRA of Avignon for their help in column construction. We are also grateful to J. M. Crumsey, K. J. Nadelhoffer and J. M. LeMoine for the English-language editing.