domain: sdv sde

text:
Julie Lucas
email: julie.lucas@univ-lr.fr

Prescilla Perrichon

Marine Nouhaud

Alexandre Audras

Isabelle Leguen

Christel Lefrancois

Aerobic metabolism and cardiac activity in the descendants of zebrafish exposed to pyrolytic polycyclic aromatic hydrocarbons

Keywords: parental transfer, pyrolytic hydrocarbons, sub-lethal concentration, metabolic rate, heart

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Aerobic metabolism and cardiac activity in the descendants of zebrafish exposed to pyrolytic polycyclic aromatic hydrocarbons

Introduction

Polycyclic aromatic hydrocarbons (PAHs) constitute an important family of persistent organic pollutants (POPs) that may be issued from natural and anthropogenic sources. Pyrolytic PAHs are released through partial combustion of organic matter at high temperature (Hylland et al. 2005) and enter in aquatic ecosystem through atmospheric deposition. These types of PAH are mainly characterized by high molecular weight PAHs (four-to six-rings) and a small proportion of alkylated compounds (e.g. methyled compounds). On the contrary, natural discharges of petroleum or oil accidents introduce petrogenic PAHs, which are dominated by low-molecular weight compounds (two-to four-rings) and alkylated compounds. In general, PAHs are characterized by their resistance and ubiquity [START_REF] Dabestani | A compilation of physical, spectroscopic and photophysical properties of polycyclic aromatic hydrocarbons[END_REF]. Found as complex mixtures in environment, they are related to multiple mechanisms of toxicity driven by their chemical properties, their concentration, and their origins. All compounds in PAHs mixture could have synergic or antagonist effects depending on their chemical properties and biotransformation [START_REF] Barron | Are aromatic hydrocarbons the primary determinant of petroleum toxicity to aquatic organisms?[END_REF]Van der Oost et al. 2003;Wassenber and Di Giulio 2004;[START_REF] Billiard | Nonadditive effects of PAHs on Early Vertebrate Development: Mechanisms and Implications for Risk Assessment[END_REF]). In addition to the risk for human health, PAHs are well described in literature especially for their carcinogenic, genotoxic and mutagenic effects on fish [START_REF] Hawkins | Use of the Japanese Medaka (Oryzias latipes) and Guppy (Poecilia reticulata) in Carcinogenesis Testing Under National Toxicology Program Protocols[END_REF][START_REF] Myers | Related Lesions and Exposure to Relationships between Hepatic Neoplasms Toxic Chemicals in Marine Fish from the U.S. West Coast[END_REF][START_REF] Tuvikene | Responses of fish to polyclic aromatic hydrocarbons[END_REF].

PAHs are also known to impact growth, reproduction and survival of aquatic organisms [START_REF] Meador | Altered growth and related physiological responses in juvenile Chinook salmon (Oncorhynchus tshawytscha) from dietary exposure to polycyclic aromatic hydrocarbons (PAHs)[END_REF][START_REF] Kim | Effects of Dietary Benzo[a]pyrene on Growth and Hematological Parameters in Juvenile Rockfish, Sebastes schlegeli (Hilgendorf)[END_REF][START_REF] Horng | A reproductive toxicology study of phenanthrene in medaka (Oryzias latipes)[END_REF]. The lipophilic property of PAHs induces their adsorption by suspended particles in aquatic ecosystem, [START_REF] Fowler | Petroleum hydrocarbons and trace metals in nearshore gulf sediments and biota before and after the 1991 war. AN assessment of temporal and spatial trends[END_REF][START_REF] Latimer | The sources, transport, and fate of Fundulus heteroclitus PAHs in the marine environment[END_REF]. Consequently, aquatic organisms may be affected by various pathways such as aqueous exposure, but also sediment contact for instance. Some studies show that PAHs have different effects on organisms. They could for instance affect the reproductive function (Collier et al., 1992;Seruto et al., 2005) through reduction or inhibition of gonads development, decrease of the eggs quality and number, as well as reduction of egg and larval viability [START_REF] Hall | Anthracene reduces reproductive potential and is maternally transferred during long-term exposure in fathead minnows[END_REF]Oris 1991, Vo n Westernhagen et al. 1981;Fletcher et al. 1982). Moreover, PAHs or their derivates could be accumulated in parent's gonads and transferred into the eggs. This precocious exposure to PAHs can be deleterious for descendants and reduce the offspring survivorship. [START_REF] Hall | Anthracene reduces reproductive potential and is maternally transferred during long-term exposure in fathead minnows[END_REF] observed that a maternal exposure to anthracene induced teratogenic effects (e.g. oedema, eyes and yolk deformities) on the progeny of fathead minnows (Pimephales promelas). PAHs-indirect effects can be also considered. Recent studies have indeed shown that maternal stress influences offspring characteristic through the production of stress hormones, such as cortisol. Level of cortisol is commonly used as an indicator of the degree of stress experienced by fish (Barton, 2002;Barton and Iwama, 1991;Wendelaar Bonga, 1997 ;Aldegunde et al., 1999;Kennedy and Farrell, 2005;Tintos et al., 2008;Marentette et al., 2013). Thereby, pollutants could disrupt physiological processes regulated by cortisol, such as metabolism or reproduction (Gesto et al., 2008;Tintos et al., 2007Tintos et al., , 2008)). In addition, maternal cortisol produced under stressor could be accumulated in eggs and affect the gametes quality (Campbell et al., 1992(Campbell et al., , 1994)), the size or the quality of the progeny of fish (McCormick 1998(McCormick , 1999(McCormick , 2006)).

In this context, the main objective of this study was to investigate the effects of pyrolytic contamination through parental transfer. The interest of this study come from the lack of investigation and understanding regarding the effect of parental PAH-exposure on the descendants. In order to do so, aerobic metabolism and cardiac frequency were measured in 5 days post fertilizations (dpf) larvae born to genitors exposed to pyrolytic-PAHs through trophic pathway. Furthermore, to have a better view of the cardiac effects in the descendants of zebrafish exposed to PAHs, genes expression in relation to heart development and activity [START_REF] Nesan | Embryo exposure to elevated cortisol level leads to cardiac performance dysfunction in zebrafish[END_REF] were measured in embryo (48 hours post-fecondation, hpf) and larvae (6dpf).

Material & Methods

Protocol of food contamination

Pyrolytic PAHs fraction (PY) has been extracted from sediment in the Seine Bay (France). According to the protocol described by [START_REF] Budzinski | Evaluation of sediment contamination by polycyclic aromatic hydrocarbons in the Gironde estuary[END_REF] and [START_REF] Cachot | Characterization of toxic effects of sediment-associated organic pollutants using the lambda transgenic medaka[END_REF], the 16 most toxicants PAHs listed by the US Environmental Protection Agency (US-EPA) have been extracted and quantified. Age-adapted food (INICIO Plus, Biomar, France) was then spiked with PY fraction using protocol adapted from [START_REF] Vicquelin | A new spiked sediment assay using embryos of the Japanese medaka specifically designed for a reliable toxicity assessment of hydrophobic chemicals[END_REF] and described in Vignet et al. (in press). The concentration 5µg.g -1 of food was considered as an environmental relevant value, since it is representative of what is measured in mollusks in the Seine bay [START_REF] Cachot | Evidence of genotoxicity related to high PAH content of sediments in the upper part of the Seine estuary (Normandy, France)[END_REF].

Based on this concentration of reference called X, three concentrations were targeted in the experimental food : 0.3X, 1X and 3X. A fourth treatment was tested for which the experimental food contained only dichloromethane, the solvent used to spiked the food. This treatment was considered as the control. PAHs compositions and concentrations in diet used for exposure are described in detail in Vignet et al. (in press). Briefly, the total concentration of PAHs (ng.g-1 dry weight food) for treatments control, 0.3X, 1X, 3X was respectively 55 ±12, 1,763 ± 0, 468, 5,816 ± 1,433, 18,151±4.983.

Zebrafish exposure

The tested progeny came from genitors directly exposed to PY-PAHs by trophic pathway. These genitors were themselves obtained from uncontaminated couples (wild-type Tuebingen strain, TU) as described in Vignet et al. (in press). Genitors were fed from the first meal (5 dpf) to the adult stage by one of the 4 types of food (i.e. control, 0.3X, 1X or 3X). These groups of contaminated individuals represented the first generation called F 0 . When they were mature (i.e. at 4 months old), some of these fish were used to form couples to obtain a new generation called F 1 . Until 5dpf, embryos and larvae F 1 were maintained in Petri dishes (6-7 ml) into ionic enriched solution of E3 at a constant temperature of 28°C and a photoperiod of 14 h light/10 h dark. Because of enough yolk sac reserves that fulfill dietary needs and the mouth opening, that occurs at the age of 5 dpf, larvae never received food before the test. The tests were made on these 5 dpf, larvae.

Metabolites quantification

PAHs metabolites were extracted from three pools of 5dpf larvae F 1 for each treatment. Larvae were homogenized in 3-4 mL of acetate buffer solution (pH= 5.0). After the addition of internal calibration compounds and beta-glucuronidase enzymes, these samples were placed at 37°C and submitted to an enzymatic deconjugation during 16 hours. Metabolites were extracted by Solid Phase Extraction (SPE) procedure using octadecyl cartridge (C 18, Bakerbond, 500 mg, 3cc, elution with 100 % of methanol) as described by [START_REF] Mazéas | Solid-phase extraction and purification for the quantification of polycyclic aromatic hydrocarbon metabolites in fish bile[END_REF]. The organic extract obtained was then concentrated under gas flow and purified by amino column (NH 2 , Supelco, 500 mg, 3cc, elution with a mix of 80/20, v/v, dichloromethane/methanol). This final extract obtained was quantified by liquid chromatography associated coupled to tandem mass spectrometry (LC/MS/MS) as described by le Dû-Lacoste (2013). PAHs metabolites in larvae are described in the Table 1.

Respirometry

Experimental set-up

The set-up consisted of 8 independent glass micro respirometer chambers (diameter d= 1.12 cm, volume V= 0.98.10 -3 l; Loligo systems, Denmark). Respirometers were submerged into buffer tanks (depth x length x height: 10x20x31 cm) and filled with oxygenated E3 solution at a constant temperature of 28°C. Peristaltic pumps controlled the water supply from the buffer tank to each respirometer. Each pump was controlled by a timer, allowing intermittent flow respirometry (Steffensen 1989), where phases of oxygen renewal alternated with phases of oxygen consumption measurements with a period of 15:15 min. Each respirometer was equipped with a fiber optic sensor (PreSens) connected to a multichannel oxygen measuring system (OXY 4 mini, PreSens) to record dissolved oxygen levels. Optic fibers were calibrated at 0% and 100% of air saturation at a temperature of 28°C. A factor of conversion based on oxygen solubility into water was used to convert oxygen data from percentage saturation to mgO 2 .l -1 (i.e. 100% was equivalent to 7.94 mgO 2 .l -1 for a 28°C temperature and a 0 salinity). Oxygen saturation was recorded every five seconds with the program Oxyview (PreSens).

Experimental protocol

Each experimental trial consisted in testing simultaneously 4 groups of 10 larvae F 1 whose genitors were exposed to one of the four treatments (0.3X, 1X, 3X and control). A total of 36 groups was tested (n control =9 groups, n 0.3X =8 groups, n 1X =12 groups, n 3X =7 groups). Each experimental trial involved two steps. First, to assess active metabolic rate (AMR), metabolism of larvae F 1 was increased through chasing [START_REF] Schurmann | Effects of temperature, hypoxia and activity on the metabolism of juvenile Atlantic cod[END_REF][START_REF] Lefrançois | Influence of ambient oxygenation and temperature on metabolic scope and scope for heart rate in the common sole Solea solea[END_REF][START_REF] Dupont-Prinet | An investigation of metabolic prioritization in the European Sea Bass, Dicentrarchus labrax[END_REF][START_REF] Cannas | PCB contamination does not alter aerobic metabolism and tolerance to hypoxia of juvenile sole (Solea solea)[END_REF][START_REF] Clark | Physiological benefits of being small in a changing world: responses of coho salmon (Oncorhynchus kisutch) to an acute thermal challenge and a simulated capture event[END_REF]. The tested groups of 10 larvae were chased with a stick into petri dishes. When exhausted (i.e. when fish did not respond to stimulation), each group was transferred into one of the 4 respirometers to assess AMR through a first oxygen consumption over the next 15 min. Second, to estimate standard metabolic rate (SMR), larvae were left undisturbed in the respirometer for 48h, during which oxygen consumption was regularly and automatically measured. After this period of measurements, larvae were removed from the respirometers and slightly anesthetized with benzocaïne at a concentration of 50mg.l -1 . The body mass of each individual was determined using a microbalance. The tested groups showed homogenous weights; the average 'group weight' being M= 0.20± 0.02 mg (mean ± S.E). Before and after each trial, a blank measurement was performed to quantify microbial oxygen consumption in the respirometer. The average of these 2 values was substracted from the measured oxygen consumption. Each group of larvae was tested once.

Calculations

Oxygen consumption MO 2 is expressed in mgO 2 .g -1 .h -1 and calculated according to the following formula:

MO 2meas =Δ[O 2 ].V. Δt -1 .M meas -1 equation (1)
where Δ[O 2 ] (in mgO 2 .l -1 ) is the variation in oxygen concentration during the measurement period Δt (in h) and V (in l) is the volume of the respirometer minus the volume of the group of 10 larvae, M meas (in g) is the mass of the corresponding tested group of 10 larvae .

An allometric relationship exists between oxygen consumption and body mass, which permitted to correct MO 2meas using the following formula:

MO 2cor =MO 2meas .(M meas .M cor -1 ) 1-b equation (2)
where MO 2cor (in mgO 2 .g -1 .h -1 ) is the oxygen consumption related to a standard fish of 1g (M cor ), MO 2meas (in mgO 2 .g -1 .h -1 ) is the oxygen consumption estimated for experimented fish whose mass was M (meas) (in g) and b is the allometric scaling exponent describing the relationship between oxygen consumption and body mass of fish.

We used b AMR =0.926 for the correction of active metabolic rate and and b SMR =0.965 for the correction of standard metabolic rate (Lucas et al., 2014).

AMR was assessed as the maximal MO 2 obtained after chasing the larvae. SMR was estimated according to the method described by [START_REF] Steffensen | Oxygen consumption in four species of teleosts from Greenland: no evidence of metabolic cold adaptation[END_REF]. Briefly, the frequency distribution of MO 2 values recorded during the last 24 hours of the test was plotted. This generally produces a bimodal frequency distribution due to the routine activity of the fish. The higher mode (i.e. the first peak, Fig. 1) is considered to reflect the SMR and the lower mode (i.e. the second peak, Fig. 1) corresponds to the routine metabolic rate (RMR), i.e. the energy required by the fish for spontaneous activity. Aerobic metabolic scope AMS was calculated as the difference between AMR and SMR. AMR, SMR and AMS were assessed for each group. The use of a microscope (Olympus SZX9), above which was placed a color camera (DMK 31AU03-The Imaging sources, Germany), permitted to record videos on IC Capture software at 15 images per second.

Videos analysis

Estimation of f H was based on the principle that each passage of blood from the atrium to the ventricle corresponds to a cardiac contraction. In transparent larvae, blood circulation between these two cardiac chambers induces a difference in contrast on which the video analysis will be based. To improve grey contrasts, Virtual

Dub software was used to convert the video in black and white through the addition of filter. Videos were recorded in bitmap format. As each movie lasted 30s and was recorded at 15 images per second, around 450 images were extracted for each video. Each of these sequences of images was then imported in the software Image J, where the use of the Plugin ROI (i.e. Region of interest) permitted to automatically delimit the atrium and the ventricle, and to calculate the alternate circulation between these two areas of interest. Data obtained were exported in Statistica for Fourrier analysis. This analysis allows assessment of cardiac frequency from the periodical movement of the heart contraction.

Cardiac mRNA expression

Transcript abundance of some genes encoding key proteins in cardiac development and activity (cardiac myosin light chain 1 (cmlc1), cardiac troponin type T2A (tnnt2a) and calcium transporting ATPase (atp2a2a) was examined in embryo (48hpf) whose genitors were exposed to 1X and control treatments and larvae F 1 (6dpf) from genitors exposed to 0.3X, 1X and control treatments.

Preparation of total RNA Fish were euthanized and frozen on liquid nitrogen prior to storage at -80°C. Total RNA from pools of 30 embryo (48hpf) or 15 larvae F 1 (6dpf) was extracted using TRIzol (Invitrogen Ltd, Cergy Pontoise, France). Five to six pools were used per treatment (0.3X, 1X and control). Due to a limited number of embryos from genitors exposed to 3X treatment, it was not possible to measure mRNA expression in these descendants. RNA was quantified by measuring the optical density at 260 nm (OD 260 ). RNA integrity was checked using the Bioanalyzer 2100 Agilent and RNA 6000 LabChip® kits (Agilent Technologies, Waldbroon, Germany)

according to the manufacturer's instructions

Real time RT-PCR

For cDNA synthesis, 1.5 µg of RNA was denatured in the presence of 0.5 µg random primers/µg RNA (Promega)

for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega), as described by the manufacturer. To inactivate the reverse transcription, samples were heated at 95°C for 10 min and then stored at -20°C. The final volume for cDNA sample was 25µl.

Control reactions were run without reverse transcriptase and used as negative control in the real-time PCR study.

Real-time RT-PCR was carried out on a StepOnePlus real time PCR system (Applied Biosystem, Courtaboeuf, France). Reactions were performed in a total volume of 10µl with 4µl diluted RT reaction, 5µl Fast SYBR-Green master Mix (Applied Biosystem, Courtaboeuf, France) and 300nM of each primer. Primer pairs are given in Table 2. A melting curve was generated to confirm product specificity. Expression levels of target genes were normalized to the geometric average of two housekeeping genes (ß-actin and ribosomal protein, L13a rpl13a).

Expression data were calculated by the 2 -∆∆Ct method.

Statistical analysis

The statistical analysis was carried out using Statistica software. For analyses regarding aerobic metabolism and cardiac frequency variables, the normality and homoscedaticity were checked through the Shapiro and Barlett tests, respectively. These conditions, necessary to apply parametric tests, were respected. A test of ANOVA was therefore used to determine significant differences due to the treatment (0.3X, 1X, 3X, control). If necessary, a Tuckey post-hoc test was applied to determine which treatments differed significantly from the control. The results were considered to be significantly different when p<0.05. Regarding cardiac mRNA expression, difference between groups and comparison of two means were assessed using Kruskal-Wallis one-way analysis of variance and Mann-Whitney U test. For all, significance was accepted at a level of 0.05.

Results

Active metabolic rate, Standard metabolic rate, Aerobic metabolic scope

Statistical analyses did not reveal any significant difference in AMR (Fig. 2A, Anova,ddl=3,F=2.46,p=0.08) and AMS (Fig. 2C, Anova, ddl=3, F=0.32, p=0.81) measured in 5dpf larvae born from genitors exposed to one of the 4 treatments tested (i.e. control, 0.3X, 1X and 3X). On the other hand, the treatment had a significant effect on SMR (Fig. 2B, Anova, ddl=3, F=32.61, p<0.01). However, post-hoc test revealed that none of the three treatments based on contaminated diet (i.e. 0.3X, 1X, 3X) differ significantly from the control (Tuckey test p>0.05 in each case). Significant differences were only observed between larvae from genitors exposed to the 3X treatment and both of the other PAH treatments (i.e. 0.3X, 1X; Tuckey test p 0.3X-3X <0.05, p 1X-3X <0.05).

Cardiac mRNA expression

The mRNA abundance of cardiac muscle genes measured in 48hpf embryo F 1 was similar between embryo from genitors exposed to 1X pyrolytic PAHs or control (Fig. 4). In 6dpf larvae F 1 , mRNA expression for myosin light chain 1 and troponin type T2A was similar in larvae F 1 whatever the diet the genitors were exposed to (Fig 5).

The mRNA expression of calcium transporting ATPase atp2a2a gene showed a different pattern. This gene was indeed upregulated in larvae F 1 whose genitors were exposed to 1X pyrolitic PAHs (Mann-Whithney U test, p<0.01). For each gene, treatments with common superscript are not significantly different

Discussion

This study carried out in zebrafish, assessed the effects of parental exposure to pyrolytic PAHs mixtures on biological parameters related to the metabolic and cardiac performance of their progeny. Regarding cardiac performance, heart rate and mRNA expression of genes encoding for cardiac activity were both modified at the environmental representative PAHs concentration 1X. On the other hand, the unique metabolic variable influenced was the SMR, but not in comparison to the control treatment.

Cardiac frequencies measured (i.e. 158.2±10.7 bt.min -1 ) for the controls are consistent with previous investigations on zebrafish. [START_REF] Barrionuevo | O 2 consumption and heart rate in developing zebrafish (Danio rerio): influence of temperature and ambient O 2[END_REF] and [START_REF] Burggren | Developing animals flout prominent assumptions of ecological physiology. Comparative biochemistry and physiology[END_REF] estimated average cardiac frequency to be 155 bt.min -1 in 5dpf larvae. The present results showed a 12.5% significant increase in cardiac frequency of larvae born from genitors exposed to the 1X PAHs treatment (Fig. 3). In addition, calcium transporting ATPase mRNA, a key cardiac gene essential for contraction [START_REF] Ebert | Calcium extrusion is critical for cardiac morphogenesis and rhythm in embryonic zebrafish hearts[END_REF], was observed to increase significantly for the same type of contamination (Fig 5). This suggests that in the progeny whose parents were exposed to the PAHs concentration 1X, heart presents an abnormal tachycardia pattern.

On the contrary, complementary work showed a similar mRNA expression of cardiac structural gene (cardiac myosin light chain 1 (cmlc1) and cardiac troponin type T2A (tnnt2a) in the progeny of parents exposed or not to PAHs treatments. This suggests a correct cardiac morphogenesis as it was observed on larvae used for cardiac frequency measurements. The pattern observed in the present study differs from recent studies investigated cardiotoxicity of PAHs in early life-stages of zebrafish directly exposed to PAHs by sediment contacts or oil.

Cardiac defects observed were indeed principally heart deformities (e.g. pericardial oedema), arrhythmia or bradycardia, and never tachycardia (Incardona et al. 2003[START_REF] Incardona | Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons[END_REF][START_REF] Incardona | Aryl hydrocarbon receptor-independent toxicity of weathered crude oil during fish de-velopment[END_REF](Incardona et al. , 2006[START_REF] Incardona | Cardiac toxicity of 5-ring polycyclic aromatic hydrocarbons is differentially dependent on the aryl hydrocarbon receptor 2 isoform during zebrafish development[END_REF][START_REF] Hicken | Sub-lethal exposure to crude oil during embryonic development alters cardiac morphology and reduces aerobic capacity in adult fish[END_REF]Perrichon, personal communication). However, comparison is limited since (i) most of these investigations have tested individual compounds at unrealistic environmental concentrations in order to trigger toxic mechanisms that impair physiological or metabolic functions [START_REF] Incardona | Aryl hydrocarbon receptor-independent toxicity of weathered crude oil during fish de-velopment[END_REF](Incardona et al. , 2006[START_REF] Incardona | Cardiac toxicity of 5-ring polycyclic aromatic hydrocarbons is differentially dependent on the aryl hydrocarbon receptor 2 isoform during zebrafish development[END_REF] and (ii) fish were directly exposed to pollutants whereas in the present study contamination was expected to occur by parental transfer.

Moreover, depending on the way of exposure, PAHs levels of contamination in organisms are expected to be different. In the present study, PAHs-metabolites concentrations measured in larvae F 1 tended to be proportional to the PAHs degree of exposure but the levels of contamination are low. Focusing on fish directly exposed, PAHs-metabolites have not been assessed in genitors at 4 months but Vignet et al., (in press) measured it on 15 dpf larvae. The concentrations tended to be proportional to the PAHs content of the food they received 9.1±0.62, 19.3±2,91, 72±11,79, 275.11±50,14 for control, 0.3X, 1X, 3X respectively. These differences in PAHsmetabolites concentrations may be explained by the fish capacity to quickly metabolize PAHs in more watersoluble components easier to excrete (Blahova et al., 2013, Dévier et al., 2013;[START_REF] Tuvikene | Responses of fish to polyclic aromatic hydrocarbons[END_REF]. Probably only a small part of these hydrocarbons derivates has been accumulated in genitor's gonads and transferred into the eggs of zebrafish, as shown in several studies in aquatic animals (Lotufo, 1998;[START_REF] Miller | Maternal transfer of organochlorine compounds in salmonines to their eggs[END_REF][START_REF] Monteverdi | Oocytic accumulation and tissue distribution of 2,3,7,8tetrachlorodibenzo-p-dioxin and benzo[a]pyrene in gravid Fundulus heteroclitus[END_REF][START_REF] Hall | Anthracene reduces reproductive potential and is maternally transferred during long-term exposure in fathead minnows[END_REF]. The lack of information regarding PAHs parental transfer in literature and these PAHs-metabolites concentrations may participate to the fact that no clear pattern regarding the effect of parental exposure to PAHs on the progeny cardiac frequency can be established.

Another hypothesis to explain the observed tachycardia pattern is that PAHs exposure could have indirect effects on cardiac activity through production of stress hormones related to the pollutant exposure, as observed in other studies (e.g. Oncorhynchus mykiss, Aldegunde et al., 1999;Tintos et al., 2008;Clupea pallasii, Kennedy and Farrell, 2005;Neogobius melanostomus, Marentette et al., 2013). In the present study, the cortisol level was not measured. However, as observed in other species, D. rerio is likely to present an elevation of the production of stress hormone induced by the exposure to pyrolytic PAHs. This cortisol increase could modulate cardiac frequency (e.g. bradychardia or tachychardia, [START_REF] Mccormick | Influence of Cortisol on Developmental Rhythms during Embryogenesis in a Tropical Damselfish[END_REF][START_REF] Nesan | Embryo exposure to elevated cortisol level leads to cardiac performance dysfunction in zebrafish[END_REF]. A relevant properties of the maternal cortisol, produced under stressor, is that it could be accumulated in eggs, affect the gametes quality (Campbell et al., 1992(Campbell et al., , 1994)), the size or the quality of the progeny of fish (McCormick 1998(McCormick , 1999(McCormick , 2006)). Based on the cortisol properties, we can suppose that this stress hormone could modulate cardiac activity and induce tachycardia in the present study. Moreover our results could be correlated with fish hyperactivity and the high level of stress, observed during behavioral challenge in other similar study (Perrichon, personal communication).

Aerobic metabolism of the progeny coming from genitors directly exposed to PAHs never differed significantly compared to control; even for the treatment 1X for which cardiac frequency was significantly influenced. Very few studies have examined influence of PAHs exposure on cardio-respiratory performance (e.g. Claireaux andDavoodi, 2010, Hicken 2011). Associated to a decrease in cardiac performance, [START_REF] Claireaux | Effect of exposure to petroleum hydrocarbons upon cardio-respiratory function in the common sole (Solea solea)[END_REF] have observed that fuel-exposed common sole displayed lower aerobic metabolism compared to control. [START_REF] Hicken | Sub-lethal exposure to crude oil during embryonic development alters cardiac morphology and reduces aerobic capacity in adult fish[END_REF] also demonstrated that early exposure to crude oil altered cardiac morphology (by modifying ventricular shape), which reduced cardio-respiratory performance and aerobic capacity in adult zebrafish. On the contrary, in the present study, variations in aerobic metabolism and cardiac frequency did not match in the larvae tested (Fig. 2 and3). This may have different explanations. First, it is worth noticing that cardiac frequency is only one cardiac parameter on which depends the oxygen transport. Taking into consideration other parameters, such as the stroke volume, should give a better overview of an organism cardiac performances. Second, the proportion of cutaneous respiration is expected to be high in early life stage, which can distort the oxygen transport capacity. Third, larvae were tested in group of 10 individuals, while cardiac frequency was measured in individuals. All these reasons may also count for the different results observed later in 2-months juveniles from the same generation F 1 for which it was observed a significant decrease in both AMR and AMS in the treatment 3X (Lucas et al., submitted). These juveniles were indeed individually tested, which allowed more acute measurements of metabolic rates.

Conclusion

The lack of notably impairments we observed does not imply that PAHs effects were absent but that the level of integration is probably finer at larval stage. Investigation on heart morphology could provide complementary information regarding these results. For instance, as these capacities depend on cardiac form and function, it could be interesting to observed atrial and ventricular shape in 5dpf larvae through specific antibodies marking on these structures. Moreover, as suggested by few studies, maternal transfer of pollutant to fish eggs and larvae could acclimatize them to toxicants, inducing potential compensations at lower level of organization [START_REF] Peake | Locomotory behavior and post-exercise physiology in relation to swimming speed, gait transition and metabolism in free-swimming smallmouth bass (Micropterus dolomieu)[END_REF][START_REF] Nye | The effect of maternal exposure to contaminated sediment on the growth and condition of larval Fundulus heteroclitus[END_REF]. In addition, it is worth noticing that cardiac frequency and metabolism was estimated in individuals, while PAHs and metabolites dosages were made on larval pools. This could participate to a mismatch in the variability related these two variables.

Fig. 1

 1 Fig. 1 An example of frequency distribution of oxygen consumption in larvae. The left and right peaks represent standard metabolic rate SMR and routine metabolic rate RMR, respectively

Fig. 2

 2 Fig. 2 Active metabolic rate (AMR, A), standard metabolic rate (SMR, B), and aerobic metabolic scope (AMS, C) of 5 dpf larvae F1 zebrafish Danio rerio coming from genitors exposed to one of the following treatments: control, 0.3X, 1X, and 3X. The environmental reference concentration X was 5.5 μg.g-1 of dry food. For SMR (B), treatment with common superscript is not significantly different

Fig. 3

 3 Fig. 3 Cardiac frequency of 5 dpf larvae F1 zebrafish Danio rerio coming from genitors exposed to one of the following treatments: control, 0.3X, 1X, and 3X. The environmental reference concentration X was 5.5 μg.g-1 of dry food. Mean values±standard deviation are plotted and treatments with common superscript are not significantly different

Fig. 4

 4 Fig. 4 Relative mRNA expression of three cardiac structural genes in 48 hpf embryo F1 zebrafish Danio rerio from genitors exposed to pyrolytic PAHs 1X treatment. Mean values ±standard deviation are plotted (N=6 pools of 30 embryo for control and N=5 for 1X treatment)

Figure 1 .

 1 Figure 1. An example of frequency distribution of oxygen consumption in larvae. The left and right peaks represent standard metabolic rate SMR and routine metabolic rate RMR, respectively.

Figure 2 .

 2 Figure2. Active metabolic rate (AMR, A), standard metabolic rate (SMR, B) and aerobic metabolic scope (AMS, C) of 5dpf larvae F 1 zebrafish Danio rerio coming from genitors exposed to one of the following treatments: control, 0.3X, 1X and 3X. The environmental reference concentration X was 5.5 µg.g -1 of dry food. For SMR (B), treatment with common superscript are not significantly different.

Figure 3 .

 3 Figure 3. Cardiac frequency of of 5dpf larvae F 1 zebrafish Danio rerio coming from genitors exposed to one of the following treatments: control, 0.3X, 1X and 3X. The environmental reference concentration X was 5.5 µg.g -1 of dry food. Mean values ±standard deviation are plotted and treatments with common superscript are not significantly different.

Figure 4 .

 4 Figure 4. Relative mRNA expression of three cardiac structural genes in 48hpf embryo F 1 zebrafish Danio rerio from genitors exposed to pyrolytic PAHs 1X treatment. Mean values ± standard deviation are plotted (N=6 pools of 30 embryo for control and N=5 for 1X treatment).

Figure 5 .

 5 Figure 5. Relative mRNA expression of three cardiac structural genes in 6dpf larvae F 1 zebrafish Danio rerio from genitors exposed to pyrolytic PAHs 0.3X and 1X treatment. The environmental reference concentration X was 5.5 µg.g -1 of dry food. Mean values ± standard deviation are plotted (N=5 pools of 15 larvae for each treatment). For each gene, treatments with common superscript are not significantly different.

Acknowledgements

We wish to thank Antoine Bonnieux, Didier Leguay, Michel Prineau, and Lucette Joassard for their help during the experiment. All experiments were carried out at Ifremer (Plateforme d'Ecophysiologie des Poissons), La Rochelle station, France. This study was financially supported by the ANR project ConPhyPoP (CES 09_002) and JL received a doctoral grant of the Regional Council of Poitou-Charentes. This study was conducted under the approval of Animal Care Committee of France under the official licence of Marie-Laure Bégout (17-010).


Anatoly Pashkevich

Roman Gomolitsky

Philippe Wenger
email: philippe.wenger@irccyn.ec-nantes.fr

Damien Chablat
email: damien.chablat@irccyn.ec-nantes.fr

CALIBRATION OF QUASI-ISOTROPIC PARALLEL KINEMATIC MACHINES: ORTHOGLIDE

Keywords: Parallel robots, calibration, parameter identification, error compensation

The paper proposes a novel approach for the geometrical model calibration of quasi-isotropic parallel kinematic mechanisms of the Orthoglide family. It is based on the observations of the manipulator leg parallelism during motions between the specific test postures and employs a low-cost measuring system composed of standard comparator indicators attached to the universal magnetic stands. They are sequentially used for measuring the deviation of the relevant leg location while the manipulator moves the TCP along the Cartesian axes. Using the measured differences, the developed algorithm estimates the joint offsets and the leg lengths that are treated as the most essential parameters. Validity of the proposed calibration technique is confirmed by the experimental results.

INTRODUCTION

Parallel kinematic machines (PKM) are commonly claimed as appealing solutions in many industrial applications due to their inherent structural rigidity, good payload-to-weight ratio, high dynamic capacities and high accuracy [START_REF] Tlusty | Fundamental Comparison of the Use of Serial and Parallel Kinematics for Machine Tools[END_REF][START_REF] Merlet | Parallel Robots[END_REF][START_REF] Wenger | Comparative study of parallel kinematic architectures for machining applications[END_REF]. However, while the PKM usually exhibit a much better repeatability compared to serial mechanisms, they may not necessarily posses a better accuracy, which is limited by manufacturing/assembling errors in numerous links and passive joints [START_REF] Wang | On the accuracy of a Stewart platform -Part I: The effect of manufacturing tolerances[END_REF]. Thus, the PKM accuracy highly relies on the accurate kinematic model, which must be carefully tuned for each manipulator separately.

Similar to the serial manipulators, the PKM calibration techniques are based on the minimization of a parameter-dependent error function, which incorporates residuals of the kinematic equations. For the parallel manipulators, the inverse kinematic equations are considered computationally more efficient (contrary to the direct kinematics, which is usually analytically unsolvable for the PKM) [START_REF] Innocenti | Algorithms for kinematic calibration of fully-parallel manipulators[END_REF][START_REF] Iurascu | Geometric algorithm for kinematic calibration of robots containing closed loops[END_REF][START_REF] Jeong | Kinematic calibration of redundantly actuated parallel mechanisms[END_REF][START_REF] Huang | A general and novel approach for parameter identification of 6-dof parallel kinematic machines[END_REF]. But the main difficulty with this technique is the full-pose measurement requirement, which is very hard to implement accurately. Hence, a number of studies have been directed at using the subset of the pose measurement data, which however creates another problem, the identifiability of the model parameters.

Popular approaches in the parallel robot calibration deal with one-dimensional pose errors using a double-ball-bar system or other measuring devices, as well as imposing mechanical constraints on some elements of the manipulator [START_REF] Daney | Kinematic Calibration of the Gough platform[END_REF]. However, in spite of hypothetical simplicity, it is hard to implement in practice since an accurate extra mechanism is required to impose these constraints. Additionally, such methods reduce the workspace size and consequently the identification efficiency.

Another category of the methods, the self-or autonomous calibration, is implemented by minimizing the residuals between the computed and measured values of the active and/or redundant joint sensors. Adding extra sensors at the usually unmeasured joints is very attractive from computational point of view, since it allows getting the data in the whole workspace and potentially reduces impact of the measurement noise. However, only a partial set of the parameters may be identified in this way, since the internal sensing is unable to provide sufficient information for the robot endeffector absolute location.

More recently, several hybrid calibration methods were proposed that utilize intrinsic properties of a particular parallel machine allowing extracting the full set of the model parameters (or the most essential of them) from a minimum set of measurements. It worth mentioning an innovative approach developed by [START_REF] Renaud | Combining end-effector and legs observation for kinematic calibration of parallel mechanisms[END_REF][START_REF] Renaud | Kinematic calibration of parallel mechanisms: a novel approach using legs observation[END_REF] who applied the vision-based measurement system for the PKM calibration from the leg observations. In this technique, the source data are extracted from the leg images, without any strict assumptions on the end-effector poses. The only assumption is related to the manipulator architecture (the mechanism is actuated by linear drives located on the base). However, current accuracy of the camera-based measurements is not high enough yet to apply this method in industrial environment.

This paper extends our previous research [START_REF] Pashkevich | Kinematic Calibration of Orthoglide-Type Mechanisms[END_REF] and focuses on the calibration of the Orthoglide-type mechanisms, which is also actuated by linear drives located on the manipulator base and admits technique of [START_REF] Renaud | Combining end-effector and legs observation for kinematic calibration of parallel mechanisms[END_REF][START_REF] Renaud | Kinematic calibration of parallel mechanisms: a novel approach using legs observation[END_REF]. But, in contrast to the known works, our approach assumes that the leg location is observed for specific manipulator postures, when the tool-center-point moves along the Cartesian axes. For these postures and for the nominal Orthoglide geometry, the legs are strictly parallel to the corresponding Cartesian planes. So, the deviation of the manipulator parameters influences on the leg parallelism that gives the source data for the parameter identification. The main advantage of this approach is the simplicity and low cost of the measuring system that can avoid using computer vision and is composed of standard comparator indicators attached to the universal magnetic stands.

The remainder of the paper is organized as follows. Section 2 describes the manipulator geometry, its inverse and direct kinematics, and also contains the sensitivity analysis of the leg parallelism at the examined postures with respect to the geometrical parameters. Section 3 focuses on the parameter identification, with particular emphasis on the calibration accuracy under the measurement noise. Section 4 contains experimental results that validate the proposed technique, while Section 5 summarizes the main contributions.

ORTHOGLIDE MECHANISM

Manipulator architecture

The Orthoglide is a three degrees-of-freedom parallel manipulator actuated by linear drives with mutually orthogonal axes. Its kinematic architecture is presented in Figure 1 and includes three identical parallel chains, which will be further referred as "legs". Each manipulator leg is formally described as PRP a R -chain, where P, R and P a denote the prismatic, revolute, and parallelogram joints respectively (Figure 2). The output machinery (with a tool mounting flange) is connected to the legs in such a manner that the tool moves in the Cartesian space with fixed orientation (translational motions). The Orthoglide workspace has a regular, quasicubic shape. The input/output equations are simple and the velocity transmission factors are equal to one along the x, y and z direction at the isotropic configuration, like in a conventional serial PPP machine (Wenger et al., 2000;[START_REF] Chablat | Architecture Optimization of a 3-DOF Parallel Mechanism for Machining Applications, the Orthoglide[END_REF]. The latter is an essential advantage of the Orthoglide architecture, which also allows referring it as the "quasi-isotropic" kinematic machine.

Another specific feature of the Orthoglide mechanism, which will be further used for the calibration, is displayed during the end-effector motions along the Cartesian axes. For example, for the x-axis motion in the Cartesian space, the sides of the x-leg parallelogram must also retain strictly parallel to the x-axis. Hence, the observed deviation of the mentioned parallelism may be used as the data source for the calibration algorithms. For a small-scale Orthoglide prototype used for the calibration experiments, the workspace size is approximately equal to 200200200 mm 3 with the velocity transmission factors bounded between 1/2 and 2 [START_REF] Chablat | Architecture Optimization of a 3-DOF Parallel Mechanism for Machining Applications, the Orthoglide[END_REF]. The legs nominal geometry is defined by the following parameters: L i = 310.25 mm, d = 80 mm, r = 31 mm where L i , d are the parallelogram length and width, and r is the distance between the points Ci and the tool centre point P (see Figure 2).

P A i  i B i C i L i d r i i j i

Modelling assumptions

Following previous studies on the PKM accuracy (Wang & Massory, 1993;[START_REF] Renaud | Combining end-effector and legs observation for kinematic calibration of parallel mechanisms[END_REF], the influence of the joint defects is assumed negligible compared to the encoder offsets and the link length deviations. This validates the following modelling assumptions: (i) the manipulator parts are supposed to be rigid bodies connected by perfect joints; (ii) the manipulator legs (composed of a prismatic joint, a parallelogram, and two revolute joints) generate a four degrees-of-freedom motions; (iii) the articulated parallelograms are assumed to be perfect but non-identical; (iv) the linear actuator axes are mutually orthogonal and intersected in a single point to insure a translational movement of the end-effector; (v) the actuator encoders are perfect but located with some errors (offsets).

Using these assumptions, there will be derived new calibration equations based on the observation of the parallel motions of the manipulator legs.

Basic equations

Since the kinematic parallelograms are admitted to be non-identical, the kinematic model developed in in our previous papers [START_REF] Pashkevich | Design Strategies for the Geometric Synthesis of Orthoglidetype Mechanisms[END_REF][START_REF] Pashkevich | Kinematic Calibration of Orthoglide-Type Mechanisms[END_REF] should be extended to describe the manipulator with different length of the legs.

Under the adopted assumptions, similar to the equal-leg case, the articulated parallelograms may be replaced by the kinematically equivalent bar links. Besides, a simple transformation of the Cartesian coordinates (shift by the vector (r, r, r) T , see Figure 2) allows to eliminate the tool offset. Hence, the Orthoglide geometry can be described by a simplified model, which consists of three rigid links connected by spherical joints to the tool centre point (TCP) at one side and to the allied prismatic joints at another side. Corresponding formal definition of each leg can be presented as PSS, where P and S denote the actuated prismatic joint and the passive spherical joint respectively.

Thus, if the origin of a reference frame is located at the intersection of the prismatic joint axes and the x, y, z-axes are directed along them, the manipulator kinematics may be described by the following equations

                x x x x x x x x x x L L e L        sin cos sin cos cos ) ( p ; (1a)                   y y y y y y y y y y L e L L        cos sin cos cos ) ( sin p ; (1b)                 e L L L z z z z z z z z z z        cos cos ) ( sin cos sin p , ( 1c
)
where p = (p x , p y , p z ) T is the output vector of the TCP position,  = ( x ,  y ,  z ) T is the input vector of the prismatic joints variables,  = ( x ,  y ,  z ) T is the encoder offset vector,  i ,  i , i{x, y, z} are the parallelogram orientation angles (internal variables), and L i are the length of the corresponding leg.

After elimination of the internal variables  i ,  i , the kinematic model (1) can be reduced to three equations It should be noted that, for the case 0

  2 2 2 2 ) ( i k j i i i L p p p         , ( 2
      z y x    and L L L L z y x   
, the nominal ""mechanical-zero"" posture of the manipulator corresponds to the Cartesian coordinates p 0 = (0, 0, 0) T and to the joints variables  0 = (L, L, L). Moreover, in such posture, the x-, yand z-legs are oriented strictly parallel to the corresponding Cartesian axes. But the joint offsets and the leg length differences cause the deviation of the "zero" TCP location and corresponding deviation of the leg parallelism, which may be measured and used for the calibration. Hence, six parameters ( x ,  y ,  z , L x , L y , L z ) define the manipulator geometry and are in the focus of the proposed calibration technique.

Inverse and direct kinematics

The inverse kinematic relations are derived from the equations (2) in a straightforward way and only slightly differ from the "nominal" case:

i k j i i i i p p L s p         2 2 2 , ( 3
)
where s

x , s y , s z { ±1} are the configuration indices defined for the "nominal" geometry as the signs of 

xp x ,  yp y ,  zp z , respectively. It is obvious that expressions (3) give eight different solutions, however the Orthoglide prototype assembling mode and the joint limits reduce this set to a single case corresponding to the s x = s y = s z = 1. For the direct kinematics, equations (2) can be subtracted pair-to-pair that gives linear relations between the unknowns p x , p y , p z , which may be expressed in the parametric form as

, ) ( 2 2 2 i i i i i i i i L t p                ( 4
)
where t an auxiliary scalar variable. This reduces the direct kinematics to the solution of a quadratic equation

2    C Bt At with the coefficients ; ) ( ) ( 2 2        j i j j i i A     x y z  x = L + L sin O p  y = L cos  z = L cos   (a) : XMax posture x y z  x = L O p  y = L  z = L (b) : Zero posture x y z  x = L -L sin O p  y = L cos  z = L cos   (c) : XMin posture
; ) ( ) ( ) ( 2 2 2 2 k k j j k j i i i i i L B                   4 / ) ( ) ( 2 4 / ) ( ) ( 2 2 4 2 2 2 k k j j k j i i i i i i i i i i L L C                                    where } , , { , , z y x k j i 
. From two possible solutions that gives the quadratic formula, the Orthoglide prototype (see Figure 1) admit a single one

A AC B B t 2 / ) 4 ( 2    
corresponding to the manipulator assembling mode.

Differential relations

To obtain the calibration equations, first let us derive the differential relations for the TCP deviation for three types of the Orthoglide postures: (i) "maximum displacement" postures for the directions x, y, z (Figure 3a); (ii) "mechanical zero" or the isotropic posture (Figure 3b); (iii) "minimum displacement" postures for the directions x, y, z (Figure 3c); These postures are of particular interest for the calibration since, in the "nominal" case, a corresponding leg is parallel to the relevant pair of the Cartesian planes.

The manipulator Jacobian with respect to the parameters  =( x ,  y ,  z ) and L = ( L x , L y , L z ) can be derived by straightforward differentiating of the kinematic equations (2), which yields

                              z z

J

It should be noted that, for the computing convenience, the above expression includes both the Cartesian coordinates p x , p y , p z and the joint coordinates  x ,  y ,  z , but only one of these sets may be treated as an independent taking into account the inverse/direct kinematic relations.

For the "Zero" posture, the differential relations are derived in the neighbourhood of the point {p 0 = (0, 0, 0) ;  0 = (L, L, L)}, which after substitution to (5) gives the Jacobian matrix

              1 0 0 1 0 0 0 1 0 0 1 0 0 0 1 0 0 1 0 J . (6)
Hence, in this case, the TCP displacement is related to the joint offsets and the leg legs variations i by trivial equations ;

i i i L p       } , , { z y x i  . ( 7
)
For the "XMax" posture, the Jacobian is computed in the neighbourhood of the point

{ ) 0 , 0 , (  LS  p ; ) , , (    LC LC LS L  ρ
}, where  is the angle between the y-, z-legs and the Xaxes:

) / sin( max L a

   ; ) ( sin    S , ) cos(   C . This gives the Jacobian                    1 1 0 1 0 0 0 1 0 0 1 0 0 1       C T T C T T x J , (8)
where ) tan(   T

. Hence, the desired equations for the TCP displacement may be written as

x x x L p       y x y x y L L T T p          1      z x z x z L C L T T p          1      (9)
It can be proved that similar results are valid for the and "ZMax" postures (differing by the indices only), and also for the "XMin", "YMin", "ZMin" postures. In the latter case, the angle  should be computed as ) / sin( min L a    .

CALIBRATION METHOD

Measurement technique

To evaluate the leg/surface parallelism, we propose a single-sensor measurement technique. It is based on the fixed location of the measuring device for two distinct leg postures corresponding to the minimum/maximum values of the joint coordinates (Figure 4). Relevant calibration experiment consists of the following steps:

Step 1. Move the manipulator to the "Zero" posture; locate two gauges in the middle of the X-leg (parallel to the axes Y and Z); get their readings.

Step 2. Move the manipulator to the "XMax" and "XMin" postures, get the gauge readings, and compute differences.

Step 3+. Repeat steps 1, 2 for the Y-and Z-legs and compute corresponding differences.

Figure 4: Measuring the leg/surface parallelism.

Calibration equations

The system of calibration equations can be derived in two steps. First, it is required to define the gauge initial locations that are assumed to be positioned at the leg middle at the "Zero" posture, i.e. at the points 2

/ ) ( i r p  , } , , { z y x i 
where the vectors r i define the prismatic joints centres: )

0 ; 0 ; ( x L x     r ; ) 0 ; ; 0 ( y L y     r ; ) ; 0 ; 0 ( z L z     r .
Hence, using the equation ( 7), the gauge initial locations can be expressed as

                   2 ; 2 ; 2 0 z z y y x x x L L L L    g                    2 ; 2 ; 2 0 z z y y x x y L L L L    g                    z z y y x x z L L L L    2 ; 2 ; 2 0 g
Afterwards, in the "XMax", "YMax", "ZMax" postures, the leg location is also defined by two points, namely, (i) the TCP, and (ii) the centre of the prismatic joint r i . For example, for the "XMax" posture, the TCP position is

) ; ; ( max        x L x LS x   p ,
and the joint position is

) 0 ; 0 ; ( max x LS L x       r .
So, the leg is located along the line

Figure 1 :

 1 Figure 1: The Orthoglide kinematic architecture. (© CNRS Photothèque / CARLSON Leif)

Figure 2 :

 2 Figure 2: Kinematics of the Orthoglide leg.

  ) which includes components of the input and output vectors p and  only. Here, the subscripts } control software (for the Orthoglide prototype,  min = -100 mm and  max = +60 mm).

Figure 3 :

 3 Figure 3: Specific postures of the Orthoglide (for the x-leg motion along the Cartesian axis X).

  a scalar parameter, [0, 1]. Since the xcoordinate of the gauge is independent of the posture, the parameter  may be obtained from some transformations, the deviations of the X-leg measurements (between the "XMax" and "Zero" postures) may be expressed as

( asin min 2     L . This system can be solved using the pseudoinverse of Moore-Penrose, which ensures the minimum of the residual square sum.

EXPERIMENTAL RESULTS

The measuring system is composed of standard comparator indicators attached to the universal magnetic stands allowing fixing them on the bases. The indicators have resolution of 10 m and are sequentially used for measuring the X-, Y-, and Z-leg parallelism while the manipulator moves between the Max, Min and Zero postures. For each measurement, the indicators are located on the mechanism base in such manner that a corresponding leg is admissible for the gauge contact for all intermediate postures (Figure 5).

For each leg, the measurements were repeated three times for the following sequence of motions: Zero  Max  Min  Zero …. Then, the results were averaged and used for the parameter identification (the repeatability of the measurements is about 0.02 mm).

To validate the developed calibration technique and the adopted modelling assumptions, there were carried out three experiments targeted to the following objectives: (#1) validation of modelling assumptions; (#2) collecting the experimental data for the parameter identification; and (#3) verification of the calibration results. The first experiment produced rather high parallelism deviation, which impels to conclude that the mechanism mechanics requires more careful tuning. Consequently, the location of the joint axes was adjusted mechanically to ensure the leg parallelism for the Zero posture.

The second experiment (after mechanical tuning) yielded lower deviations, twice better than for the first experiment. For these data, the developed calibration algorithm was applied for three sets of the model parameters: for the full set {, L} and for the reduced sets {} and {L}. As follows from the identification results (Table 1), the algorithms is able to identify simultaneously both the joint offsets and  and the link lengths L. However, both  and L (separately) demonstrate roughly the same influence on the residual reduction, from 0.32 mm to 0.14 mm, while the full set {, L} gives further residual reduction to the 0.12 mm only. This motivates considering  as the most essential parameters to be calibrated. Accordingly, the identified vales of the joint offsets were input into the control software.

The third experiment demonstrated good agreement with the expected results. In particular, the average deviation reduced down to 0.15 mm, which corresponds to the measurement accuracy. On the other hand, further adjusting of the model to the new experimental data does not give the residual reduction.

Hence, the calibration results confirm validity of the proposed identification technique and its ability to tune the joint offsets and link lengths from observations of the leg parallelism. Other conclusion is related to the modelling assumption: for further accuracy improvement it is prudent to generalize the manipulator model by including parameters describing the orientation of the prismatic joint axes, i.e. relaxing assumption (iv) (see sub-section 2.2).

CONCLUSIONS

This paper proposes further developments for a novel calibration approach for parallel manipulators, which is based on observations of manipulator leg parallelism with respect to some predefined planes. This technique employs a simple and low-cost measuring system composed of standard comparator indicators, which are sequentially used for measuring the deviation of the relevant leg location while the manipulator moves the TCP along the Cartesian axes. From the measured differences, the calibration algorithm estimates the joint offsets and the link lengths that are treated as the most essential parameters to be tuned. The validity of the proposed approach and efficiency of the developed numerical algorithm were confirmed by the calibration experiments with the Orthoglide prototype, which allowed essential reduction of the residuals and corresponding improvement of the accuracy.

Future work will focus on the expanding the set of the identified model parameters, their identifiably analysis, and compensation of the non-geometric errors.


Anatoly Pashkevich

Damien Chablat
email: damien.chablat@irccyn.ec-nantes.fr

Philippe Wenger
email: philippe.wenger@irccyn.ec-nantes.fr

Philippe Wenger Kinematic

Kinematic Calibration of the Orthoglide-Type Mechanisms

Keywords: parallel robots, kinematic calibration, model identification, joint offsets, and error compensation

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

Parallel kinematic machines (PKM) are commonly claimed to offer several advantages over serial manipulators, like high structural rigidity, better payload-to-weight ratio, high dynamic capacities and high accuracy [START_REF] Merlet | Parallel Robots[END_REF][START_REF] Tlusty | Fundamental Comparison of the Use of Serial and Parallel Kinematics for Machine Tools[END_REF]. As stated by a number of authors [START_REF] Tsai | Robot Analysis: the Mechanics of Serial and Parallel Manipulators[END_REF][START_REF] Wenger | A comparative study of serial and parallel mechanism topologies for machine tools[END_REF], the conventional serial kinematic structures have already achieved their performance limits, which are bounded by high component stiffness required to support sequential joints, links and actuators. Thus, the PKM are prudently considered as promising alternatives to their serial counterparts that offer faster, more flexible, more accurate and less costly solutions. However, while the PKM usually demonstrate a much better repeatability compared to serial mechanisms, they may not necessarily posses a better accuracy, which is limited by manufacturing/assembling errors in numerous links and passive joints [START_REF] Wang | On the accuracy of a Stewart platform -Part I: The effect of manufacturing tolerances[END_REF]. Besides, for the non-Cartesian parallel architectures, some kinematic parameters (such as the encoder offsets) cannot be determined by direct measurement. These motivate intensive research on PKM calibration.

Similar to the serial manipulators, the PKM calibration procedures are based on the minimisation of a parameter-dependent error function, which incorporates residuals of the kinematic equations. For the parallel manipulators, the inverse kinematic equa-tions are considered computationally more efficient, since the most of the PKM admits a closed-form solution of the inverse kinematics [START_REF] Innocenti | Algorithms for kinematic calibration of fully-parallel manipulators[END_REF][START_REF] Iurascu | Geometric algorithm for kinematic calibration of robots containing closed loops[END_REF][START_REF] Huang | A general and novel approach for parameter identification of 6-dof parallel kinematic machines[END_REF]. But the main difficulty with the inverse-kinematics-based calibration is the full-pose measurement requirement, which is very hard to implement accurately [START_REF] Jeong | Kinematic calibration of redundantly actuated parallel mechanisms[END_REF] Recently, several hybrid calibration methods were proposed that utilize intrinsic properties of a particular parallel machine allowing to extract the full set of the model parameters (or the most essential of them) from a minimum set of the pose error measurements. An innovative approach for the I4 parallel mechanism has been developed by [START_REF] Renaud | Combining end-effector and legs observation for kinematic calibration of parallel mechanisms[END_REF][START_REF] Renaud | Kinematic calibration of parallel mechanisms: a novel approach using legs observation[END_REF] who applied vision to perform measurement on manipulator legs, which connect the end-effector to the base-mounted linear actuators. This paper focuses on the Orthoglide-type mechanisms, which kinematic architecture admits parallel leg motions (along the longitudinal axis). From observations the leg parallelism at prespecified postures, the proposed calibration algorithm estimates the joint offsets that are treated as the most essential parameters to be adjusted. The main advantage of this approach is the simplicity and low cost of the measuring system composed of standard comparator indicators attached to the universal magnetic stands. They are sequentially used for measuring the deviation of the relevant leg location while the manipulator moves the TCP along the Cartesian axes.

The remainder of the paper is organised as follows. Section 2 describes the manipulator geometry, its inverse and direct kinematics, and also contains the sensitivity analysis of the leg parallelism at the examined postures with respect to the joint offsets. Section 3 focuses on the measurements and parameter identification. Section 4 contains experimental results and their discussion, while Section 5 summarises the main results.

KINEMATIC MODEL

Manipulator geometry

The Orthoglide is a three degree-of-freedom mechanism actuated by three linear drives with mutually orthogonal axes [START_REF] Chablat | Architecture Optimization of a 3-DOF Parallel Mechanism for Machining Applications, the Orthoglide[END_REF]. Its kinematic architecture is presented in Fig. 1 and includes three identical parallel chains that are formally described as PRPaR, where P, R and Pa denote the prismatic, revolute, and parallelogram joints.

The mechanism input is made up by three actuated orthogonal prismatic joints. The output machinery (with a tool mounting flange) is connected to the prismatic joints through a set of three kinematic chains that further referred as manipulator "legs". The legs are made with articulated parallelograms that are oriented in a manner that the output body is restricted to the translational movements only.

A specific feature of the Orthoglide mechanism, which will be further used for the calibration, is displayed during the end-effector motions along the Cartesian axes. For example, for the x-axis motion in the Cartesian space, the sides of the x-leg parallelogram must also retain strictly parallel to the x-axis. Hence, the observed deviation of the mentioned parallelism may be used as the data source for calibration algorithms.

Modelling assumptions

Following previous studies on the parallel mechanism accuracy (Wang, andMassory, 1995, Renaud, et al., 2004;[START_REF] Caro | Sensitivity Analysis of the Orthoglide, a 3-DOF Translational Parallel Kinematic Machine[END_REF], the influence of the joint/link defects is assumed negligible compared to the joint positioning errors mainly caused by the encoder offsets. This validates the following modelling assumptions:

(i) the manipulator parts are supposed to be rigidbodies connected by perfect joints; (ii) the articulated parallelograms are assumed to be identical and perfect, which insure that their sides stay parallel in pares for any motions; (iii) the manipulator legs (composed of one prismatic joint, one parallelogram, and three revolute joints) are identical and generate a five-DOF motion each; (iv) the linear actuator axes are considered mutually orthogonal and intersected in a single point to insure a translational three-DOF movement of the end-effector.

The actuator encoders are assumed to be perfect but their location (zero position) is defined with some errors that are treated as the offsets to be estimated. Using these assumptions, there will be developed a convenient calibration methodology based on the observation of the parallel gel motions.

Basic equations

Under the above assumptions, the Orthoglide can be presented by a simplified model, which consists of three rod-links connected by spherical joints to the tool centre point (TCP) at one side and to the corresponding prismatic joints at another side. Thus, if the origin of a reference frame is located at the intersection of the prismatic joint axes and the x, y, z-axes are directed along them, the manipulator geometry may be described by the equations

[ ] 2 2 2 2 ) ( L p p p z y x x = + + ρ Δ + ρ - [ ] 2 2 2 2 ) ( L p p p z y y y x = + ρ Δ + ρ - + (1) [ ] 2 2 2 2 ) ( L p p p z z z y x = ρ Δ + ρ - + +
where p = (p x , p y , p z ) is the output position vector, ρ = (ρ x , ρ y , ρ z ) is the input vector of the joints variables, Δρ = (Δρ x , Δρ y , Δρ z ) is the encoder offset vector, and L is the length of the parallelogram principal links. Hence, within the adopted model, four parameters (Δρ x , Δρ y , Δρ z , L) define the manipulator geometry, but because of the rather tough manufacturing tolerances, the leg length is assumed to be known and only the joint offsets (Δρ x , Δρ y , Δρ z ) are in the focus of the proposed calibration technique.

It should be noted that for the nominal ''mechanicalzero'' posture corresponding to the joints variables ρ 0 = (L, L, L), the x-, y-and z-legs are oriented strictly parallel to the relevant Cartesian axes. But the joint offsets cause the deviation of the "zero" location and corresponding deviation of the leg parallelism with respect to the manipulator base surface. The latter may be (i) measured and (ii) computed applying the direct kinematic algorithm for the joint variables ρ = (L+Δρ x , L+Δρ y , L+Δρ z ). However, the capability of this simple technique is limited by evaluating the offset of the z-axis encoder only, since the Orthoglide mechanical design does not allow making similar measurements for the remaining pairs of the legs, with respect to the xz-and yz-planes.

Inverse and direct kinematics

To derive calibration equations, first let us expand some previous results on the Orthoglide kinematics [START_REF] Pashkevich | Design Strategies for the Geometric Synthesis of Orthoglide-type Mechanisms[END_REF] taking into account the encoder offsets. The inverse kinematic relations are derived from the equations (1) in a straightforward way and only slightly differ from the "nominal" case

x z y x x x p p L s p ρ Δ - - - + = ρ 2 2 2 y z x y y y p p L s p ρ Δ - - - + = ρ 2 2 2 (2) z y x z z z p p L s p ρ Δ - - - + = ρ 2 2 2
where s x , s y , s z ∈ { ±1} are the configuration indices defined for the "nominal" manipulator as signs of (ρ x -p x ) , (ρ y -p y ), and (ρ z -p z ) respectively. It is obvious that expressions (2) define eight different solutions to the inverse kinematics, however the Orthoglide assembling and joint limits reduce this set for a single case corresponding to the s x = s y = s z = 1.

For the direct kinematics, the equations (1) can be subtracted pair-to-pair that gives the following expression for the unknowns p x , p y ,

p z } , , { ; 2 z y x i t p i i i i i ∈ ρ Δ + ρ + ρ Δ + ρ = ( 3
)
where t is an auxiliary scalar variable. This reduces the direct kinematics to the solution of a quadratic equation

0 2 = + + c bt at with the coefficients ; ) )( ( ∏ ≠ ρ Δ + ρ ρ Δ + ρ = j i j j i i a ∏ ρ Δ + ρ = i i i b 2 ) ( ; ; 4 ) ( 2 2 ∑ - ρ Δ + ρ = i i i L c } , , { , z y x j i ∈
From two possible solutions that give the quadratic formula, the Orthoglide prototype (see Fig. 1) admits only one expressing as

2 ( 4 ) / 2 t b m b ac a = -+ - .

Sensitivity analysis

To evaluate the encoder offset influence on the legs parallelism with respect to the Cartesian planes XY, YZ, and YZ, let us derive the differential relations for the TCP deviation for three types of the Orthoglide postures: mechanical zero" posture and "maximum/minimum displacement" postures for the directions x, y, z. These postures are of particular interest for the calibration since in the "nominal" case (no encoder offsets) at least one leg is parallel to the corresponding pair of the Cartesian planes.

The desired differential relations may be derived from the Orthoglide Jacobian, the inverse of which is obtained from (1) as

1 1 ( , ) 1
1 y z x x x x x z y y y y y x z z z z p p p p p p p p p p p p ρ ρ ρ ρ ρ ρ - ⎡ ⎤ ⎢ ⎥ - - ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ = - - ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ - - ⎣ ⎦ J p ρ (4)
For the "mechanical zero" posture, the differential relations are derived in the neighbourhood of the point p 0 = (0, 0, 0) and ρ 0 = (L, L, L), which gives the identity Jacobian matrix

0 0 3 3 ( , ) × = J p ρ I .
Hence, in this case the TCP displacement is related to the joint offsets by trivial equations ;

i i p ρ Δ = Δ } , , { z y x i ∈
. Taking into the account the Orthoglide geometry, this deviation may be estimated by evaluating parallelism of the legs with respect to the Cartesian planes. How-ever, as mentioned in sub-section 2.3, this technique is feasible for z-direction only and may produce estimation of Δρ z merely.

For the "maximum displacement" posture in the xdirection, the differential relations are derived in the neighbourhood of the point ) 0 , 0 , sin ( α = L p and ) , , (

α α α + = LC LC LS L ρ
, where, α is the angle between the y-, z-legs and corresponding Cartesian axes, and

α = α = α α cos ; sin C S
. This gives the inverse Jacobian as a lower triangle matrix

1 0 0 ( , ) 1 0 0 1 T T α α ⎡ ⎤ ⎢ ⎥ = ⎢ ⎥ ⎢ ⎥ ⎣ ⎦ J p ρ (5) where α = α tan T
. Hence, the differential equations for the TCP displacement may be written as ;

; ; z x z y x y x x T p T p p ρ Δ + ρ Δ = Δ ρ Δ + ρ Δ = Δ ρ Δ = Δ α α (6)
and the joint offset influences on the TCP deviation is estimated by factors 1.0 and T α . It is also worth mentioning that measurement of the x-leg parallelism with respect to the XY-plane gives an equation for estimating the offset Δρ x (provided that the offset Δρ z has been obtained from the "mechanical zero").

Similar results are valid for the "maximum displacement" postures in the y-and z-directions (differing by the indices only), and also for the "minimum displacement" postures. In the latter case, the angle α should be computed as

) ( asin min L ρ = α
. Hence, the leg parallelism is rather sensitive to the joint offsets and relevant deviations Δp x , Δp y , Δp z , and may be used for the offset identification.

CALIBRATION METHODOLOGY

Measurement technique

To identify the model parameters, we propose a single-sensor measurement techniques for the leg/surface parallelism (Fig. 2). It is based on fixed location of the measuring device for two distinct leg postures corresponding to the minimum/maximum of the joint coordinates. Relevant calibration experiment consists of the following steps:

Step 1. Move the manipulator to the "mechanical zero"; locate two gauges in the middle of the X-leg (parallel to the axes Y and Z); get their readings.

Step 2. Move the manipulator to the "X-maximum" and "X-minimum" postures, get the gauge readings, and compute differences

- - + + Δ Δ Δ Δ x x x x z y z y , , , .
Step 3+. Repeat steps 1, 2 for the Y-and Z-legs and compute corresponding differences.

Calibration equations

The calibration equations can be derived using expressions from sub-section 2.5. First, it is required to define the gauge initial location. For the X-leg, it is the midpoint of the line segment bounded by the TCP (Δρ x , Δρ y , Δρ z ) and the centre of the X-joint (L+Δρ x , 0, 0). This yields the following expression for the X-leg midpoint: ( 2 ; 2; 2)

x y z L ρ ρ ρ + Δ Δ Δ .
For the "X-maximum" posture, the X-leg location is also defined by same two points, but their coordinates are ) ; ; (

z x y x x T T LS ρ Δ + ρ Δ ρ Δ + ρ Δ ρ Δ + α α α and ) 0 ; 0 ; ( x LS L ρ Δ + + α
respectively. Then, it may be written the equations of a straight-line passing along the X-leg and computed the point corresponding to

x L x ρ Δ + = 2
. Hence, finally, the deviations of the X-leg measurements may be expressed as

z x x y x x S T S z S T S y ρ Δ + ρ Δ + = Δ ρ Δ + ρ Δ + = Δ α α α + α α α + ) 5 . 0 ( ; ) 5 . 0 ( (7)
Similar approach may be applied to the "Xminimum" posture, as well as to the equivalent postures for the Y-and Z-legs. This gives the system of twelve linear equations in three unknowns and the angles α 1 , α 2 are computed for the maximum and minimum postures respectively. The reduced version of this system may be obtained if to assess the leg/plane parallelism by the difference between the "maximum" and "minimum" postures. The latter leads to the system of six linear equations in three unknowns.

⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎣ ⎡ Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ = ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ ρ Δ ρ Δ ρ Δ ⋅ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢

EXPERIMENTAL RESULTS

Experimental setup

The measuring system is composed of standard comparator indicators attached to the universal magnetic stands allowing fixing them on the manipulator bases. The indicators have resolution of 10 μm and are sequentially used for measuring the X-, Y-, and Z-leg parallelism while the manipulator moves between the Max, Min and Zero postures. For each measurement, the indicators are located on the mechanism base in such manner that a corresponding leg is admissible for the gauge contact for all intermediate posters (Fig. 3).

For each leg, the measurements were repeated three times for the following sequence of motions: Zero → Max → Min → Zero→ …. Then, the results were averaged and used for the parameter identification. It should be noted that measurements demonstrated very high repeatability (about 0.02 mm).

Calibration results

The first calibration experiment produced rather high parallelism deviation, up to 2.37 mm, which in the frames of the adopted kinematic model was expected to be reduced down to 1.07 mm only (Table 1). The corresponding residual norm was also rather high and yielded unrealistic estimate of the measurement noise parameter σ ≈ 1.0 mm. It impels to conclude that the mechanism mechanics requires more careful tuning. Consequently, the location of the joint axes was adjusted mechanically to ensure the leg parallelism for the Zero posture.

Fig. 3. Experimental Setup

The second calibration experiment (after mechanical tuning) yielded lower parallelism deviations, less than 0.70 mm. For these data, the developed calibration algorithm yielded the joint offsets that are expected to reduce the deviation down to 0.28 mm. Besides, the estimated value of σ ≈ 0.28 mm is rather realistic taking into account both the measurement accuracy and the manufacturing/assembling tolerances. Accordingly, the identified vales of the joint offsets were input into the control software.

The third experiment (validation of the identified parameters) demonstrated good agreement with the expected results. In particular, the maximum parallelism deviation reduced down to 0.34 mm (expected 0.28 mm), while its root-mean-square decreased down to 0.21 mm (expected 0.20 mm). On the other hand, further adjusting of the kinematic model to the new experimental data gives both negligible improvement of the r.m.s. and rather small alteration of the model parameters. It is evident that further reduction of the parallelism deviation is bounded by the manufacturing and assembling errors.

Hence, the calibration results confirm validity of the proposed identification technique and its ability to tune the joint offsets from observations of the leg parallelism.

CONCLUSIONS

This paper proposes a novel calibration approach for the parallel manipulators, which is based on observations of manipulator leg parallelism with respect to some predefined planes. Presented for the Orthoglide-type mechanisms, this approach may be also applied to other manipulator architectures that admit parallel leg motions along the longitudinal axis.

The proposed calibration technique employs a simple and low-cost measuring system composed of standard comparator indicators attached to the universal magnetic stands. They are sequentially used for measuring the deviation of the relevant leg location while the manipulator moves the TCP along the Cartesian axes. From the measured differences, the calibration algorithm estimates the joint offsets that are treated as the most essential parameters to be tuned. The validity of the proposed approach and efficiency of the developed numerical algorithm were confirmed by the calibration experiments with the Orthoglide prototype, which allowed reducing the residual r.m.s. by three times.

To increase the calibration precision, future work will focus on the development of the specific assembling fixture ensuring proper location of the linear actuators and also on the essential theoretical issues such as calibration experiment planning, expanding the set of the identified model parameters and their identifiably analysis, and compensation of the nongeometric errors non detected within the frames of the adopted kinematic model.

Fig. 1 .

 1 Fig. 1. The Orthoglide mechanism

Fig. 2 .

 2 Fig. 2. Measuring the leg/surface parallelism

Table 1 .

 1 Experimental data and expected improvements of accuracy(model-based)

	Data Source	Δx y mm	Δx z mm	Δy x mm	Δy z mm	Δz x mm	Δz y mm	r.m.s. mm
			Initial settings (before mechanical tuning and calibration)
	Experiment #1	+0.52	+1.58	+2.37	-0.25	-0.57	-0.04	1.19
	Expected improvement	-0.94	+0.63	+1.07	-0.84	-0.27	+0.35	0.74
				After mechanical tuning (before calibration)
	Experiment #2	-0.43	-0.37	+0.42	-0.18	-1.14	-0.70	0.62
	Expected improvement	-0.28	+0.25	+0.21	-0.14	-0.13	+0.09	0.20
					After calibration
	Experiment #3	-0.23	+0.27	+0.34	-0.10	-0.09	+0.11	0.21
	Expected improvement	-0.29	+0.23	+0.25	-0.17	-0.10	+0.08	0.20


Mathieu Luglia
email: mathieu.luglia@imbe.fr

Stéven Criquet

Max Sarrazin

Fabio Ziarelli

Daniel Guiral

Avicennia germinans and Laguncularia racemosa located in urban context in French Guiana (Montabo's bay, Cayenne). This mangrove results from HydroEco2013 -4 th International Multidisciplinary Conference on Hydrology and Ecology Mangrove soils Who drives their recycling functions?

The study site is a fringing mangrove containing a mixed-species assemblage of

Introduction

The Amazon River is responsible of a huge discharge of sediments in Atlantic Ocean. A portion of 10 to 20 % of this suspended particulate matter is diverted and follows a northwestern path under the influence of Guiana Current that results in a spectacular migration of 1 600 km of subtidal mud belt along the coastline of Guianas to the Orinoco Delta. In the intertidal zone, mud banks form extensive mud flats whose periodic dewatering by tide cycles progressively generates the stabilization of the upper intertidal part. Its colonization by biofilms and then by propagules imported from fringing mature mangroves, promotes extending of mangrove ecosystem. In such environment, highly constrained by a rapid and intensive hydro-sedimentary dynamic, the successional dynamic of mangrove ecosystem gradually leads to the development of vegetation stage series, from pioneers to matures and sometimes to senescents. The interactions between sediment, organic matter (OM), aboveground vegetation and meioand macro-fauna are responsible of changes in ground characteristics and pore-water composition. These are related to microbial community functioning which is a central component of biogeochemical processes and nutrient dynamics that take place in mangrove ecosystem.

Objectives

Conclusion

In pioneer stages, the microbial communities are fuelled and controlled by low contents and poor quality of soil TOC. Thus, they show low activities and are constrained to diversify their C-acquisition mechanisms resulting in high functional diversities. As mangrove succession proceeds inward, a TOC enrichment occurs which results in higher microbial activities. Concomitantly, an increased imprint of mangrove vegetation on SOM quality is revealed. These differences in organic sources and qualities lead to functional loss and specialization of rhizospheric microbial communities and subsequently, evidence an enhancement of above-belowground functional linkages.

  Our objectives were to understand which environmental factors were the main drivers of the expression of microbial functions in these intertidal sediments. With this aim, we distinguished several classes of environmental factors potentially involved in regulation of microbial functions: Mangrove evolutionary stages defined by 3 facies, i.e. pioneer (P, <1y), coppice (C, <5y) and young forest (F, <10y); Hydro-climatic conditions, i.e. two contrasted seasons: wet (WS) and dry (DS); Hydro-and pedochemical properties of soils, including qualitative characterization of soil OM (SOM) by SS 13 C CP-MAS NMR spectroscopy; Rhizospheric effects. Functions of microbial communities investigated were: • Microbial respiration (MR, Oxitop®); • Potential catabolic richness and diversity by community level physiological profiling (CLPP, Biolog EcoPlates TM ); • Activity of 6 enzymes involved in CNPS biogeochemical cycles. Types of soils investigated were: • Within P facies (3): a non-rhizospheric soil (NRZ), rhizospheric soils of Avicennia (RZA) and (RZL); • C facies (1): an undifferentiated rhizospheric soil (RZ); • F facies (1): RZ soil.

Functional

  Plume of suspended particulate matter from Amazon's estuary (left) and its north-western migration along the coastline of French Guiana (right) Laguncularia racemosa located in urban context in French Guiana (Montabo's bay, Cayenne). This mangrove results from a massive arrival of silt, which has begun to deposite since 2003, where 3 main facies occur corresponding to vegetation evolutionary stages. Early colonization stage by micro-phytobenthos and mangrove of the upper intertidal part of mud flat (Montabo's bay, january 2006) properties of soils Mangrove structure and plot characteristics Spatial and seasonal variations SOM enrichment as succession proceeds; Complex salinity and nutrient fluctuations due to: hydro-climatic variation, microtopography, ocean-distance, vegetation cover and evapotranspiration, redox status (S 2-); C/N ratio around 10 are representative of and micro-phytobenthos derived OM (Marchand et al., 2003; Debenay et al., 2007); Progressive enrichment of soils in alkyl C and carbohydrate C indicated an increased influence of mangrove inputs on SOM quality. (n = 3) standard deviations. IVI: Importance Value Index (Curtis & McIntosh, 1950) Biplot of PCA of hydro-pedochemical properties of soils Symbols depict means (n = 5) with standard deviations (in brackets) on PC1 and PC2 Vector scale: x3; C/N: molar C/N ratio; OlsP: Olsen phosphorus; totP: total phosphorus; TOC: total organic carbon; S2-: sulphide; totS: total sulphur Values are means (n = 5) standard deviations or are based on a composite sample. For each season, values followed by the same letters in a column do not differ significantly from each other at P < 0.05 (one-way ANOVA, test of multiple comparisons of Tukey). Humification index: Alkyl C to O-alkyl C ratio Rapid and intensive erosion phase of mangrove (Montabo's bay, april 2011)

PC2

  5 -2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0 Enzyme activities and microbial respiration are higher;Functional diversity shows an inverse pattern than enzymes; No significant rhizospheric effect on functional patterns of P facies soils;Functional patterns are maintained between the 2 hydro-climatic seasons.As succession proceeds…Strongincrease of microbial respiration in relation with TOC enrichment; increases are those of lipase and β-glucosidase activities, which are highly correlated with increased availability of their respective natural substrates, i.e. plant lipids and carbohydrates (e.g. cellulose, wax, cutin); PCA map of CLPPs exhibits a functional specialization of microbial communities toward some C sources, i.e. methylenic structures vs forest carbohydrate polymers vs pioneer community. been supported by the Programme Biodiversité Marine of the Direction de l'Environnement, de l'Aménagement et du Logement (DEAL) from French Guiana, and by the Institut de Recherche pour le Développement (IRD) from France. Biplot of PCA of enzyme and catabolic activities, and functional diversities of soils Symbols depict means (n = 5) with standard deviations (in brackets) on PC1 and PC2 Vector scale: x3; H': Shannon's diversity index; MR: microbial respiration; FDA: fluorescein diacetate hydrolase; β-glu: β-glucosidase; Lip: lipase; AcP: acid phosphomonoesterase; Ure: urease; ArylS: arylsulphatase Biplot of PCA of catabolic profiles of soils performed on 27 substrates Symbols depict means (WS: n = 10, DS: n 15) with standard deviations (in brackets) on PC1 and PC2 Vector scale: x3; only the most driving variables are depicted; B3: acid; C1: tween 40; D1: tween 80; E1: α-cyclodextrin; E4: L-threonine; F1: glycogen; H4: putrescine Microbial respiration and lipase and β-glucosidase activities of soils Bars depict means (n = 5) with standard deviations (in brackets). For each season, bars within a graph followed by the same letters or numbers do not differ significantly from each other at P < 0.05 (one-way ANOVA, test of multiple comparisons of Tukey). U: µmole hydrolyzed substrate.min-1 Pearson's correlation coefficients (r). *** depicts a significance level of P < 0