Marc Metian
email: mametian@gmail.com

Paco Bustamante

Laetitia Hédouin

M Metian A-B

Dr Michel Warnau
email: m.warnau@iaea.org

Accumulation of nine metals and one metalloid in the tropical scallop Comptopallium radula from coral reefs in New Caledonia

Keywords: tropical environment, trace elements, arsenic, radiotracers, Pectinidae, risk assessment, bioindicator species

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Accumulation of nine metals and one metalloid in the tropical scallop Comptopallium radula from coral reefs in New Caledonia

Introduction

The urban and industrial expansion in tropical coastal zones increases the release of contaminants which can constitute a threat to local marine ecosystems and globally affect marine diversity [START_REF] Peters | Ecotoxicology of tropical marine ecosystems[END_REF]. The SW lagoon of New Caledonia (South Pacific Ocean) represents a tropical case study as it is subject to large inputs of heavy metals mainly due to intense land-based mining activities [START_REF] Ambastian | High metals inputs to closed seas: the New Caledonia lagoon[END_REF]). New Caledonia is the third largest producer of nickel (Ni) in the world and metal contamination mainly concerns Ni and its mining by-product such as cobalt (Co), chromium (Cr) and manganese (Mn) which occur at elevated concentrations in Ni ores. Furthermore, the city of Noumea produces sewage sludge, which can also lead to metal contamination in the surrounding lagoon. Nevertheless, published information about the contamination status of the New Caledonia marine environment is extremely scarce [START_REF] Labrosse | New Caledonia[END_REF] and limited to a narrow range of species [START_REF] Bustamante | Bioaccumulation of 12 trace elements in the tissues of the nautilus Nautilus macromphalus from New Caledonia[END_REF][START_REF] Hédouin | Nickel bioaccumulation in bivalves from the New Caledonia lagoon: Seawater and food exposure[END_REF][START_REF] Monniot | Cellular sites of iron and nickel accumulation in ascidians related to the naturally and anthropic enriched New Caledonian environment[END_REF]. Environmental studies are therefore needed to understand the behaviour and fate of metals in this area in order to develop a program of coastal zone monitoring and improve local marine resource management.

The use of biomonitor species to examine metal contamination is a powerful tool to reveal the bioavailability of the considered contaminants (e.g. [START_REF] Rainbow | Cosmopolitan biomonitors of trace metals[END_REF]. In this respect, bivalves have been extensively used as biomonitor species because of their high capacity to bioaccumulate various contaminants (see e.g. [START_REF] Goldberg | Emerging problems in the coastal zone for the twenty-first century[END_REF][START_REF] Rainbow | Biomonitoring of heavy metal availability in the marine environment[END_REF].

Among bivalves, scallops have been shown to concentrate various trace elements to a large extent (e.g. [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus (L.) and Chlamys opercularis (L.)[END_REF][START_REF] Bustamante | Subcellular and body distributions of 17 trace elements in the variegated scallop Chlamys varia from the French coast of the Bay of Biscay[END_REF][START_REF] Uthe | Cadmium in sea scallop (Plactopecten magellanicus) tissues from clean and contaminated areas[END_REF], even in remote areas such as Antarctica that are non subject to direct anthropogenic inputs [START_REF] Berkman | Trace metal concentrations in scallops around Antarctica: Extending the Mussel Watch Programme to the Southern Ocean[END_REF][START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF][START_REF] Viarengo | Cu, Zn, Cd content in different tissues of the Antarctic scallop Adamussium colbecki (Smith 1902): role of metallothionein in the homeostasis and in the detoxification of heavy metals[END_REF].

Within the huge bivalve stock in New Caledonia, exhibiting around 1 200 species among the whole 8 000 bivalve species estimated in the world [START_REF] Bouchet | Combien y a-t-il de mollusques en Nouvelle-Calédonie?[END_REF], 30 pectinid species have been described to date [START_REF] Dijkstra | Pectinidés des fonds meubles dans les lagons de Nouvelle-Calédonie et des Chesterfield. 2ème partie: systématique et biogéographie[END_REF]). In the SW lagoon, Comptopallium radula appears relatively common and constitutes, with Mimachlamys gloriosa and Brachtechlamys vexillum the major species targeted for human consumption. Whereas information on stock, reproduction and taxonomy of these scallops is available [START_REF] Dijkstra | Pectinidés des fonds meubles dans les lagons de Nouvelle-Calédonie et des Chesterfield. 2ème partie: systématique et biogéographie[END_REF][START_REF] Lefort | Growth and mortality of the tropical scallops: Annachlamys flabellata (Bernardi), Comptopallium radula (Linne) and Mimachlamys gloriosa (Reeve) in South-West Lagoon of New Caledonia[END_REF], to the best of our knowledge, no study on the bioaccumulation of trace elements in the soft tissues of New Caledonian scallops has been published to date.

The aim of this study was therefore to examine the bioaccumulation and tissue distribution of a wide range of trace elements in the scallop C. radula using both laboratory experiments under controlled conditions and in situ investigations. Firstly, the uptake kinetics of 4 selected radiotracers ( 109 Cd, 57 Co, 54 Mn, and 65 Zn) and their body distribution were determined following seawater exposure. Secondly, 1 metalloid (As) and 9 metals (Ag, Cd, Co, Cr, Cu, Fe, Mn, Ni, and Zn) were analysed in the tissues of scallops from reference and contaminated sites in order to evaluate the bioaccumulation capacities of C. radula and its potential as a biomonitor species for trace element contamination. Finally, the human risk link to scallop consumption is discussed.

Material and Methods

Radiotracer experiments

Twenty scallops (Comptopallium radula [Linnée, 1758]) were collected in the vicinity of Noumea City (i.e. Sainte Marie Bay) by SCUBA diving in October 2004. The organisms (76 ± 3 mm length) were experimentally exposed to four radiotracers ( 109 Cd, 57 Co, 54 Mn, and 65 Zn) via seawater. Seawater was spiked with low nominal activities of each selected radiotracer: 1.76 kBq l -1 109 Cd, 0.91 kBq l -1 57 Co, 0.65 kBq l -1 54 Mn,, 0.65 kBq l -1 65 Zn.. In terms of stable metal concentration, these additions corresponded to 9 10 -3 ng Cd l -1 , 6 10 -3 ng Co l -1 , 5 10 -2 ng Mn l -1 and, 4 10 -3 ng Zn l -1 , which are 1 to 5 orders of magnitude lower than the background concentrations of these metals in open seas [START_REF] Bruland | Trace elements in seawater[END_REF].

The uptake of the radiotracers was followed during four days using gamma spectrometric techniques. Uptake kinetics were expressed as change in concentration factor (CF; ratio between activity of the radiotracer in the whole soft tissues or in a body compartment -Bq g -1 wet wt-and time-integrated activity of radiotracer in seawater -Bq g -1 -) according to time.

Radiotracer uptake kinetics were described using a simple linear regression model

CF t = k u t
where CF t is the concentration factor at time t (d) and k u is the uptake rate constant (d -1 ) [START_REF] Whicker | Radioecology: nuclear energy and the environment[END_REF].

At various time intervals (12 h, 30 h, 48 h, 72 h, and 96 h), four individuals were randomly sampled and dissected in order to determine the tissue distribution of the elements. Shells, digestive gland, kidneys, gills, gonad, adductor muscle and the remaining soft tissues were separated and counted using a high-resolution γ-spectrometry system consisting of 4 coaxial Ge (N-or P-type) detectors (EGNC 33-195-R, Canberra ® and Eurysis ® ) connected to a multichannel analyzer and a computer equipped with a spectra analysis software (Interwinner ® 6).

The radioactivity of the samples was determined by comparison with standards of known activities and appropriate geometries and was corrected for background and physical decay of the radiotracers. Counting times were adapted to obtain counting rates with relative propagated errors lower than 5%.

Trace element analyses

Ten scallops were collected by SCUBA diving in October 2004 in the southern New Caledonian lagoon in two bays showing different physico-chemical specificities (Fig. 1): Maa Bay (n=5; 76 ± 6 mm length) and Sainte Marie Bay (n=5; 77 ± 2 mm length). This last site, located to the East of Noumea, is characterized by elevated inputs of sewage sludge [START_REF] Jaquet | Impact des apports en nutriments sur le réseau trophique planctonique du lagon sud-ouest de la Nouvelle-Calédonie[END_REF]. Moreover terrigenous inputs due the open casting mining are released by the Coulée River and deposited along the shoreline, reaching areas as distant as Sainte Marie Bay [START_REF] Fernandez | A combined modelling and geochemical study of the fate of terrigenous inputs from mixed natural and mining sources in a coral reef lagoon (New Caledonia)[END_REF]. Conversely, the Maa Bay is located 20 km North-West of Noumea and is considered to be exempt from direct anthropogenic inputs of metals and the area is sparsely inhabited. Moreover, the Maa Bay is not surrounded by lateritic soils (viz. a typical source of metal contamination by natural erosion) and is therefore considered as a noncontaminated "reference" site in this study.

In order to verify the contamination level of the two locations, sediments were collected by SCUBA diving in parallel to organisms. Sediments were stored in acid-washed and hermetically sealed plastic bag until return to the laboratory.

Upon arrival to the laboratory, organisms were depurated for 24 h in clean seawater aquaria to remove pseudo-faecal and faecal material from the digestive tract. Each individual was measured and weighed prior to dissection: the digestive gland, kidneys, gills, gonads and adductor muscle were carefully removed from each individual. The remaining tissues were also taken into account in order to calculate the whole metal content of the organisms.

All samples (scallop tissues and sediments) were dried for several days at 60°C until they reached a constant weight. Then, sediments were sieved on a 1 mm meshed sieve in order to eliminate heterogeneous materials (e.g. stones, fragment of corals) prior to elemental analysis.

Aliquots of the samples ranging from 50 to 300 mg were digested using a 3:1 (v:v) nitrichydrochloric acid mixture with 65% HNO 3 (Merck, suprapur quality) and 70% HCl (Merck, suprapur quality). Acidic digestion was performed overnight under ambient temperature and then heated in a microwave during 30 min with increasing temperature until 105°C, and 15 min at 105°C (1 200 W). After the mineralization process, each sample was diluted to 30 or 50 ml with milli-Q quality water, according to the volume of acid added to the mineralization (3 ml or 4.5 ml).

Ag, As, Cd, Co, Cr, Cu, Fe, Mn, Ni and Zn were analysed either by ICP-OES (Varian ® Vista-Pro) or ICP-MS (Varian ® Ultra Mass 700). Reference tissues (dogfish liver DOLT-3, NRCC, and lobster hepatopancreas TORT-2, NRCC) were treated and analysed in the same way as the samples. Results were in good agreement with the certified values, and the standard deviations were low, proving good repeatability of the method. The results for standard reference materials displayed recoveries of the elements ranging from 79% to 122% (n=10).

For each set of analyses, blanks were included in each analytical batch. The detection limits (µg g -1 dry wt) for ICP-OES were 10. 1 (As),0.8 (Cr),0.5 (Cu),0.3 (Fe),0.04 (Mn),1.1 (Ni) and 0.7 (Zn) and for ICP-MS, they were 0.1 (Ag), 0.3 (Cd), 0.03 (Co). All metal concentrations are given on a dry weight basis (µg g -1 dry wt).

Preliminary risk assessment for consumers

A maximum recommended consumption of scallops was evaluated on the basis of the Provisional Maximum Tolerable Daily Intake (PMTDI) or Provisional Tolerable Weekly Intake (PTWI) given by the Joint Expert Committee on Food Additives (http://www.inchem.org/pages/jecfa.html).

In this calculation, metal sources supplied by other meals or by drinking water in the same day were not taken into account, i.e. only metal intake coming from the scallop meal has been considered. The PMTDI for Cu, Fe, and Zn are respectively 500, 800 and 1 000 µg kg -1 d -1 and PTWI for As and Cd are 15 and 7 µg kg -1 wk -1 (JECFA 2006;WHO 1989). The calculation was done for As, Cd, Cu, Zn and Fe by dividing the load (µg) of each element measured in whole soft parts or in the adductor muscle (for both sites) by the respective PMTDI or PTWI and by a consumer average body weight (viz. 50 kg and 80 kg body weight for female and male human, respectively). In this manner, results indicated the maximum number of consumed C. radula not to exceed. Although C. radula is generally eaten whole, both whole soft parts and the adductor muscle alone were considered in the risk calculations for comparison purpose (viz., the adductor muscle is the only tissue eaten in the most usually marketed scallop species).

Data treatment

Statistical analyses of the data were performed by 1-or 2-way ANOVA, followed by the multiple comparison test of Tukey. The variability explained by each factor was derived from the sum of squares [START_REF] Warnau | Distribution of heavy metals in the echinoid Paracentrotus lividus from the Mediterranean Posidonia oceanica ecosystem: seasonal and geographical variations[END_REF]. After verification of the variance homogeneity, 2way analysis of variance (ANOVA) was used with sampling location and body compartment as fixed factors. When the homogeneity hypothesis was rejected by ANOVA, a multiple comparison test of Tukey was carried out to assess the most significant impact levels in the examined stations, and then the most significant metal concentrations in the body compartments. Comparison of data expressed as percentages (body distribution) was carried out after arcsine transformation of the data in respect with basic normality requirements of parametric tests [START_REF] Zar | Biostatistical analysis[END_REF].

Concerning radiotracer data analyses, kinetics of metal uptake in scallops were fitted using linear regression routines (Statistica ® 6). The best fit of the different regressions was selected by examination of residuals and R 2 . Linearity of the kinetics was tested by the linearity test for regression with replication [START_REF] Zar | Biostatistical analysis[END_REF].

The level of significance for statistical analyses was always set at α = 0.05.

Results

Laboratory experiments: radiotracer uptake study

The bioaccumulation kinetics of the 4 radiotracers in the whole soft parts of C. radula were best fitted by a linear model (Fig. 2). The uptake rate constants (k u ) found for the studied radiotracers rank as follows: Mn = Co < Cd < Zn. After 4 days of exposure, the soft-parts reached concentration factors (CFs) of 62 ± 14 for Cd, 15 ± 8 for Co, 15 ± 4 for Mn, and 149 ± 34 for Zn (Fig. 2). In addition, CFs were calculated for each organ (Table 1). The latter ones clearly show the contrasting efficiencies of metal accumulation by scallop tissues. Indeed, the highest CF was found in the kidneys for 109 Cd (391 ± 69) and 57 Co (603 ± 298) and in the digestive gland for 65 Zn (549 ± 92). In the case of the 54 Mn, the CF of gills, kidneys and digestive gland approximately reached the same value (~30) after 4d of exposure.

At the end of the exposure period, the distribution of the radiotracers was compared among the scallop body compartments following two steps (Table 2): 1) between the shell and the soft parts 2) among the different tissues and organs constituting the soft parts. Considering the whole scallops, 57 Co and 54 Mn were mainly (i.e. 92 and 96%, respectively) contained in the shell. Among the soft tissues, 54 Mn was mainly associated with the digestive gland (33%) and the gills (33%), whereas kidneys contained most of the 57 Co (65%). The two other radioisotopes were mainly present in the soft parts (69% for 109 Cd and 58% for 65 Zn), with 65% of the soft part 65 Zn activity being in the digestive gland whereas 109 Cd was mainly present in the gills (38%) and the digestive gland (25%).

In situ analyses

Arsenic and metal concentrations in the sediment and in the tissues and organs of C. radula collected from the two sampling sites are given in Tables 3 and4. Except for Ag which concentrations were below the detection limit, sediments from Maa Bay displayed significantly higher elemental concentrations compared to the supposedly more contaminated site of Sainte-Marie Bay (Table 3). In contrast, scallops from Maa Bay showed higher concentrations of As, Cd, and Fe in their tissues whereas Ag was far more concentrated in scallops from Sainte-Marie Bay. Independently of the sampling site, the digestive gland and kidneys of C. radula generally showed the highest element concentrations (Table 4). The distribution patterns of elemental concentrations were similar between scallops from both sampling sites except for Ag which specifically accumulated in the digestive gland in Maa Bay and in the kidneys in Sainte Marie Bay.

The body distribution of the different elements among the tissues and organs of the scallops from both sites are compared in Figure 3. The digestive gland clearly exhibited the highest proportion of Ag, As, Cr, Fe and Zn in both areas. In the case of Cd and Ni, the digestive gland and kidneys contained most of the metal burden. Regarding Co and Cu, the major part of the metals was contained in kidneys in Sainte-Marie Bay whereas in Maa Bay, the digestive gland also stored a large fraction of Co and contained the major part of Cu. In the case of Mn, distribution among tissues and organs is rather homogeneous in scallops from the two bays.

Risk assessment

The element content in the whole soft parts of the scallops allowed the computation of the number of bivalves to be eaten by male and female Human to reach the PMTDI or, in the case of As and Cd, the PTWI (Table 5). Overall, As appears as the main element of concern regarding the consumption of C. radula from both locations since the consumption of 4 (for women) or 8 (for men) scallops would result in exceeding the PTWI threshold. For the other elements, the consumption of more than 40 scallops is necessary to reach the recommended PMTDI or PTWI.

When only the adductor muscle was considered, the associated risk was lower as expected (element concentrations in the muscle were always lower than in whole soft tissues). If any, As was the only element of concern; however 27 adductor muscles for women and 44 for men should be eaten before reaching the PTWI threshold (data not shown). For the other elements, the consumption of more than 374 scallops is necessary to reach the recommended PMTDI or PTWI.

Discussion

Several studies have pointed out the ability of various scallop species to accumulate high levels of trace elements in their tissues (e.g., [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus (L.) and Chlamys opercularis (L.)[END_REF][START_REF] Bustamante | Subcellular and body distributions of 17 trace elements in the variegated scallop Chlamys varia from the French coast of the Bay of Biscay[END_REF][START_REF] Uthe | Cadmium in sea scallop (Plactopecten magellanicus) tissues from clean and contaminated areas[END_REF]. Such a bioaccumulation ability does not appear being linked to specific anthropogenic contamination of the environment since scallops from remote areas such as the North Atlantic or the Antarctica were shown to contain elevated Cd concentrations compared to related or even identical species from temperate regions [START_REF] Berkman | Trace metal concentrations in scallops around Antarctica: Extending the Mussel Watch Programme to the Southern Ocean[END_REF][START_REF] Bustamante | Interspecific and geographical variations of trace element concentrations in Pectinidae from European waters[END_REF][START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF][START_REF] Viarengo | Cu, Zn, Cd content in different tissues of the Antarctic scallop Adamussium colbecki (Smith 1902): role of metallothionein in the homeostasis and in the detoxification of heavy metals[END_REF]). Conversely, very few studies have focused on tropical scallops [START_REF] Francesconi | Cadmium in the saucer scallop, Amusium balloti, from Western Australian water: concentrations in adductor muscle and redistribution following frozen storage[END_REF]) and the current literature globally lacks related baseline information.

In this work, the bioaccumulation efficiency of trace elements of the tropical scallop Comptopallium radula has been evaluated by comparing individuals collected from two contrasting coastal areas. To assess the difference in the contamination status of both stations, trace elements were analysed in sediments (Table 3). Surprisingly, element concentrations in sediment were high in the reference site (Maa Bay) compared to those from the site close to Noumea City and subject to higher terrigenous inputs (Sainte-Marie Bay). The only exception to this was Ag which was below the detection limit of the method whereas Co, Cr, Cu, Mn, Ni and Zn concentrations were at least two times higher in the sediment from Maa Bay compared to Sainte-Marie Bay. Even if these results are somewhat surprising, they demonstrate that the two sampling sites are actually contrasting from the point of view of the relative bioavailability of the former elements.

Regarding the levels in C. radula, scallop tissues highly concentrated the analysed elements irrespective of the environment, which is consistent with reported data for other pectinid species (e.g., [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus (L.) and Chlamys opercularis (L.)[END_REF][START_REF] Bustamante | Interspecific and geographical variations of trace element concentrations in Pectinidae from European waters[END_REF][START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF][START_REF] Uthe | Cadmium in sea scallop (Plactopecten magellanicus) tissues from clean and contaminated areas[END_REF]. Interestingly, bioconcentration ability of C. radula allowed measuring relatively high levels of elements, even when sediment analysis showed concentrations below detection limit, as it was the case for Ag (see Tables 3 and4). Out of the 10 analysed elements, Ag levels were significantly higher (p<0.0001) in C. radula collected in Sainte-Marie Bay whereas As, Cd and Fe concentrations were higher in scallops from Maa Bay; the other elements did not show significant differences (p>0.05) between the two bays.

Silver is a proxy for anthropogenic input in coastal waters due to its high enrichment in sewage sludge from coastal cities [START_REF] Andren | Silver in the environment: transport, fate and effects[END_REF][START_REF] Luoma | Fate, bioavailability and toxicity of silver in estuarine environments[END_REF]. Hence higher levels of Ag in scallop tissues from Sainte-Marie Bay indicated that this area is subject urban pollution which is supporting the results of the modelling approach recently published by [START_REF] Fernandez | A combined modelling and geochemical study of the fate of terrigenous inputs from mixed natural and mining sources in a coral reef lagoon (New Caledonia)[END_REF]. Specifically, exposure to high Ag levels in Sainte Marie Bay lead to the accumulation of the metal in the kidneys of C. radula rather than in the digestive gland as observed in Maa Bay. In other Pectinidae species, the digestive gland generally exhibits the highest Ag concentrations among soft tissues [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus (L.) and Chlamys opercularis (L.)[END_REF]Bustamante andMiramand 2004, 2005;[START_REF] Segar | The distribution of the major and some minor elements in marine animals. Part II. Molluscs[END_REF]. To the best of our knowledge, these very different Ag storage strategies of C. radula between two sites have never been reported for the Pectinidae family and deserve further investigation.

In contrast to Ag, As has received little attention in pectinids. The levels of this element in the soft parts of C. radula were quite high in both bays (i.e. 45 ± 6 and 87 ± 12 µg g -1 dry wt in Sainte-Marie Bay and Maa Bay, respectively) which rather contrasts with published data in Pectinidae from temperate zones which display generally much lower concentrations [START_REF] Bustamante | Subcellular and body distributions of 17 trace elements in the variegated scallop Chlamys varia from the French coast of the Bay of Biscay[END_REF][START_REF] Lai | Arsenic speciation in scallops[END_REF]. For example, the variegated scallop Chlamys varia from the Bay of Biscay (France) reached maximum As concentrations of 25 ± 5 µg g -1 dry wt in the whole soft parts [START_REF] Bustamante | Subcellular and body distributions of 17 trace elements in the variegated scallop Chlamys varia from the French coast of the Bay of Biscay[END_REF]. Bivalves generally display a limited capacity in accumulating As from seawater (e.g. [START_REF] Hédouin | Caractérisation d'espèces bioindicatrices pour la surveillance des activités minières et la gestion de l'environnement en milieu récifal et lagonaire: application au lagon de la Nouvelle-Calédonie[END_REF][START_REF] Ünlü | Factor affecting the flux of arsenic through the mussel Mytilus galloprovincialis[END_REF] and available literature indicates that the high As levels measured in tropical pectinids are probably originating from their diet (see e.g. [START_REF] Neff | Ecotoxicology of arsenic in the marine environment[END_REF]). Hence, higher As levels in C. radula soft tissues could indicate a high dietary source and/or an elevated bioavailability of As in their environment. Such an hypothesis would be consistent with coastal organic contamination due to agricultural activities and waste discharges from shrimp aquaculture that has been reported for the two bays where C. radula was collected [START_REF] Labrosse | New Caledonia[END_REF][START_REF] Lemonnier | Effect of water exchange on effluent and sediment characteristics and on partial nitrogen budget in semi-intensive shrimp ponds in New Caledonia[END_REF]. Indeed, important discharges of N-enriched compounds could locally modify the N:P ratio and as phytoplankton metabolises As more easily in a phosphate-depleted environment (e.g. [START_REF] Smedley | A review of the source, behaviour and distribution of arsenic in natural waters[END_REF], this might result in As transfer to filter-feeders and enhanced As bioaccumulation in their tissues [START_REF] Benson | Arsenic accumulation in Great Barrier Reef invertebrates[END_REF]. Similarly, the higher As concentrations observed in scallops from Maa Bay that in Sainte Marie Bay would likely to be due to difference in dietary bioavailability of As between the two areas.

Higher Cd and Fe concentrations in C. radula from Maa Bay also support this hypothesis. Indeed, authors generally agree that high Cd concentrations in scallop tissues are related to their intense feeding rates (e.g. [START_REF] Berkman | Trace metal concentrations in scallops around Antarctica: Extending the Mussel Watch Programme to the Southern Ocean[END_REF][START_REF] Palmer | Trace metal concentrations in two shellfish species of commercial importance[END_REF] and Fe tends to complex with organic matter or colloids, leading to a high bioavailability to phytoplankton [START_REF] Chen | Marine diatom uptake of iron bound with natural colloids of different origins[END_REF]. Other studies also reported similar results with higher As and/or Cd concentrations in scallop from clean sites compared to contaminated ones (viz. C. varia from France, [START_REF] Bustamante | Subcellular and body distributions of 17 trace elements in the variegated scallop Chlamys varia from the French coast of the Bay of Biscay[END_REF] Placopecten magellanicus from Canada, [START_REF] Uthe | Cadmium in sea scallop (Plactopecten magellanicus) tissues from clean and contaminated areas[END_REF]. In both cases, these atypical levels were attributed to feeding and nutritional inadequacy of the diet. A further characterization of food composition (stomach content analyses) and/or metal concentrations in the food of the scallop in the sampling areas would be the next step to validate such an assumption. Even if the food seems to be the major accumulation pathway, the contribution of dissolved compartment cannot be neglected. Indeed, radiotracer experiments revealed the relatively high uptake of waterborne 109 Cd in scallops, clearly showing that the high accumulation of Cd would not be only restricted to dietary inputs.

Although not different between the two studied stations, Co, Cr and Ni concentrations measured in the tissues of C. radula appear relatively elevated when compared with data from the literature [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus (L.) and Chlamys opercularis (L.)[END_REF][START_REF] Bustamante | Subcellular and body distributions of 17 trace elements in the variegated scallop Chlamys varia from the French coast of the Bay of Biscay[END_REF][START_REF] Pesch | Trace metals in scallops from within and around two ocean disposal sites[END_REF]. Some of the variations could account for the specific characteristics of metal accumulation, but high Co, Cr and Ni concentrations might rather be due to natural and anthropogenic inputs of these metals in the context of the local geology of New Caledonia since lateritic soils are particularly rich in these 3 very elements [START_REF] Labrosse | New Caledonia[END_REF]. However, the lack of differences in tissues between the two sampling sites was unexpected in regards to sediment results and suggests that the scallops readily accumulate these three metals until a threshold level above which a higher contamination does not affect any longer the levels within C. radula tissues. Field experiments such as transplantations from relatively clean to strongly contaminated sites would be interesting to validate this hypothesis.

Studies on metal concentrations in the tissues of scallops from various locations around the world have highlighted their common ability to concentrate Mn and Zn in the kidneys up to very high levels, typically exceeding 1 000 µg g -1 dry wt (see Table 6). However, C. radula does not follow this trend (Table 4), and rather behaves as the southern and the northern scallops Adamussium colbecki and Patinopecten yessoensis, respectively [START_REF] Ishii | Determination and distribution of trace elements in marine invertebrates[END_REF][START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF]). Although kidney cells of A. colbecki exhibit the same cytoarchitecture as the other Pectinidae, it has been shown that the low renal bioaccumulation capacity for Mn and Zn in this species was due to the low degree of mineralization of their nephrolithes, viz. the renal phosphate-rich concretions that are well known for their ability to trap and detoxify intracellular metals [START_REF] George | Isolation and elemental analysis of metal-rich granules from kidney of the scallop Pecten maximum (L.)[END_REF][START_REF] Nigro | Ultrastructural localization of metal binding sites in the kidney of the Antarctic scallop Adamussium colbecki[END_REF]). Hence, the atypical low levels of Mn and Zn in C. radula kidneys might be due to interspecific diversity in the amount and/or degree of mineralization of renal concretions.

The lower Zn accumulation capacity of the kidneys compared to the digestive gland was also observed in our laboratory experiment: 65 Zn concentration factor was far higher in the digestive gland than in kidneys of C. radula. Interestingly, [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus (L.) and Chlamys opercularis (L.)[END_REF] showed that the Queen scallop Aequipecten opercularis (a scallop presenting typical, highly mineralized renal concretions) concentrated 65 Zn to a much higher extent in kidneys than in digestive gland.

These observations would support the hypothesis that C. radula might have developed an alternative detoxification strategy for Zn in the digestive gland to compensate the supposedly low mineralization of its renal concretions.

In New Caledonia, C. radula is commonly fished and consumed whole. Therefore, we performed a preliminary risk assessment taking into account the trace element concentrations measured in scallops from the two selected bays. Accordingly, As appears as the only limiting element for human consumption of these scallops. Indeed, consumption of the whole soft parts of 4 to 8 C. radula during a single week would lead to exceed the PTWI value recommended for As by WHO (viz. 15 µg kg -1 wk -1 ; WHO 1989). However, one has to keep in mind that As speciation has not been taken into account in the present computation. Now, it is well known that both bioavailability and toxicity of As largely depend on its chemical speciation (see e.g. [START_REF] Neff | Ecotoxicology of arsenic in the marine environment[END_REF][START_REF] Warnau | Arsenic: Is it worth monitoring in the Mediterranean Sea? Marine Sciences and Public Health -Some Major Issues[END_REF]). Inorganic As species (i.e. arsenite and arsenate), on which is based the PTWI, are the most toxic forms both in terms of acute toxicity and carcinogenicity whilst it is generally reported that marine organisms bioaccumulate the element mainly as organic (e.g., arsenobetaine and arsenosugars) compounds that are not or slightly toxic (ibid.). Since our preliminary computations were made using the concentrations of total As, it is obvious that the actual number of scallops needed to reach As PTWI will be higher than 4 (for women) to 8 (for men). However, it has been shown in several marine species that inorganic As can sometimes represents a high proportion of total As concentrations (see e.g. [START_REF] Fattorini | Arsenic speciation in tissues of the Mediterranean polychaete Sabella spallanzanii[END_REF][START_REF] Warnau | Arsenic: Is it worth monitoring in the Mediterranean Sea? Marine Sciences and Public Health -Some Major Issues[END_REF]).

Hence, rather than speculating on these values, the present computations should be considered as a warning signal about the very possible threat due to eating large meals composed of these scallops. Although traditional habits are not easily changed, a solution could be to recommend the sole consumption of adductor muscles (as is the case for most other pectinid species) since results showed consuming muscle was a non-risk issue, even without taking into account As speciation, as a minimum of 27 muscles would be needed to reach the As PTWI.

Conclusions

This work has shown that C. radula could be used to biomonitor Ag contamination in the New Caledonian lagoon. For the other elements being accumulated differently between the two sampling sites (i.e. As, Cd, and Fe), the differences between sites seem to reflect the elemental variations in the scallop environment, supporting the use of C. radula as a valuable biomonitor species for As, Cd and Fe contamination. However, additional information has to be collected from the field to understand more precisely the reason of such a difference. Furthermore, the contribution of the different routes of intake (seawater vs food) needs to be characterised. In this respect, a set of laboratory experiments using radiotracer techniques has been planned to explore the "dietary contamination pathway" hypothesis. Our results also allowed speculating on the occurrence of different accumulation/detoxification strategies of various metals (i.e. Ag, Mn and Zn) within the Pectinidae family. Finally, a preliminary estimation of trace element exposure to humans through scallop consumption has warned consumers about the possible health risks linked to elevated As accumulation by C. radula.

  Figure 1. Sampling locations along the SW coast of New Caledonia.

Figure 2 .

 2 Figure 2. Whole-body uptake kinetics of 109 Cd, 57 Co, 54 Mn and 65 Zn in Comptopallium radula

Figure 3 .

 3 Figure 3. Comparison of trace element distribution (mean % ± SD; dry wt basis; n=5) in

  Fig.1

  Fig.3

Table 1 .

 1 Concentration factor (mean ± SD; n=4) of the radiotracers in the different body compartments of Comptopallium radula after 4 d of seawater exposure.

	Compartment	109 Cd	57 Co	54 Mn	65 Zn
	Digestive gland	107 ± 29	9.7 ± 4.1	29 ± 11	549 ± 92
	Kidneys	391 ± 69	603 ± 298	33 ± 12	184 ± 52
	Gonad	76 ± 75	3.1 ± 1.8	14 ± 6	55 ± 14
	Muscle	7.0 ± 1.9	0.6 ± 0.3	2.0 ± 0.4	18 ± 5.3
	Gills	142 ± 29	3.2 ± 0.5	24 ± 5.3	96 ± 25
	Remaining tissues	3.2 ± 0.6	0.2 ± 0.1	0.6 ± 0.2	4.2 ± 0.9

Table 2 .

 2 Metal distribution (mean % ± SD, wet wt basis; n=4) between shell and soft parts; and within the soft parts of Comptopallium radula after 4 d of seawater exposure.

	Compartment	109 Cd	57 Co	54 Mn	65 Zn
	Shell	31 ± 6	92 ± 4	96 ± 1	42 ± 9
	Soft parts	69 ± 6	8 ± 4	4 ± 1	58 ± 9
	Digestive gland	25 ± 7	9.2 ± 2.4	33 ± 7	65 ± 3
	Kidneys	13 ± 1	81 ± 4.9	5.6 ± 1.7	3.2 ± 0.6
	Gonad	14 ± 11	2.5 ± 0.6	14 ± 4	5.8 ± 1.2
	Muscle	8.3 ± 1.0	2.8 ± 1.2	12 ± 1	11 ± 1.9
	Gills	38 ± 7	3.9 ± 1.7	33 ± 7	13 ± 1.8
	Remaining tissues	2.2 ± 0.3	0.8 ± 0.5	2.2 ± 0.4	1.5 ± 0.3

Table 3 .

 3 Element concentrations (mean ± SD; µg g -1 dry wt; n=5) in sediment from the two sampling locations.

		Maa Bay		Sainte-Marie Bay
	Ag		< dl*			< dl*
	As	6.4 ± 0.3	5.8 ± 0.2
	Cd	1.0 ± 0.2		< d.l.*
	Co	4.6 ± 2.3	1.9 ± 0.1
	Cr	44	±	8	15	±	1
	Cu	11	±	3	0.8 ± 0.1
	Mn	132 ±	8	33	±
	Ni	64	± 13	12	±	1
	Zn	15	±	3	3.9 ± 0.1
	*dl : detection limit

Table 4 .

 4 Element concentrations (mean ± SD and min-max values, µg g -1 dry wt; n=5) in tissues and organs of the scallop Comptopallium radula from two stations in the South-West lagoon of New Caledonia.

	Ag	As	Cd	Co	Cr	Cu	Fe	Mn	Ni	Zn

Acknowledgements. The IAEA is grateful for the support provided to its Marine Environment Laboratories by the Government of the Principality of Monaco. Authors gratefully thank O.

Pringault (IRD-Noumea) for his help during the fieldwork and C. Churlaud (CCA, Univ. La Rochelle) for her participation in the analytical process. MW is an Honorary Senior Research Associate of the National Fund for Scientific Research (NFSR, Belgium). This work was supported by the IAEA, the CRELA (Univ. La Rochelle) and the PNEC Programme (Chantier Nouvelle-Calédonie).


Claude-Eric Eric Parveaud

Johanna Brenner

Sophie-Joy Ondet

Christelle Gomez

Gilles Libourel

François Warlop

Laurent Brun

Vincent Mercier

Guy Clauzel

Jean-Marc Marc Audergon

Gilles Ubourel '

Assessment of diseases susceptibility of peach cultivars in experimental plots and on-farm for organic and low-input systems. Baseline of french case studies

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

Montfavet Cedex, France

The choice of the cultivar is one of the keystones to reduce pesticide input in peach orchards. Breeders have selected peach cultivars that have mainly been assessed on yield and fruit quality in conventional farming systems. The disease susceptibility was not considered as a key criterion. Despite a great turn-over of new peach cultivars, their suitability for organic and low-input systems remains unknown for most of

  them. A first program has been carried out from 2001 to 2008 in order to assess peach leaf curl, powdery mildew and aphid susceptibilities of 28 cultivars in a farm network and experimental stations. A high variability of leaf curl and powdery mildew susceptibilities was observed among the cultivars. ln 2009, the two most promising cultivars have been included in a second assessment program among 10 other cultivars. A randomized experimental plot design took into account the spatial distribution of the disease pressure. The variability of peach leaf curl susceptibility between cultivars was strongly influenced by the disease pressure. Since -2011, sùsceptibility to peach leaf curi, aphids, blossom and Coryneum blight of 10 established peach cultivars has been assessed in a network of commercial organicallv farmed plots. Methodological considerations include the interaction between disease epidemiology, design and management of the orchard and the observed cultivar responses to specifie pathogens. The potential and limits of farmers' implication in cultivar assessment is discussed. Keywords: cultivar susceptibility, fruit quality, Taphrina deformans, Monilia sp., Sphaerotheca pannosa var. persicae, organic farming.


domain: sdv info

text:
Thi Mong Diep Nguyen

S Alves

S Métayer-Coustard

C Praud

P Froment

E Blesbois

S12 Effects of the AMP-Kinase Modulators AICAR, Metformin and Compound C on chicken spermatozoa viability, motility and acrosome reaction

Spermatozoa are highly specialized cells whose main properties include motility and acrosome reaction. These processes require a high level of energy related to the intracellular ATP / 5'AMP ratio. AMPK (5'-AMP activated protein kinase) is a sensor of this ratio and its numerous roles include regulation of glucose, lipid, and protein metabolisms. When AMPK is activated, it stimulates catabolic pathways that produce ATP and simultaneously inhibits ATPconsuming anabolic pathways that adjust the cellular energy balance. In the study, we investigated the intracellular localization of AMPK in chicken spermatozoa using immunofluorescence and examined the effects of AMPK activators AICAR and Metformin (Met) or inhibitor Compound C (CC) on spermatozoa motility and ability to achieve the acrosome reaction. Our results show that AMPK protein is expressed in the acrosome, midpiece and flagellum of spermatozoa. Spermatozoa motility (CASA) and ability to accomplish acrosome reaction were inhibited by CC without effect on viability (Sybr14/PI). At the opposite, Met or AICAR stimulated the motility and the ability to achieve the acrosome reaction. These results show that AMPK stimulation may improve some essential functions of chicken spermatozoa.

Introduction

Sperm functions are critically controlled through the phosphorylation state of specific proteins. The 5'-AMP activated protein kinase (AMPK) is a serine/threonine protein kinase that is a sensor of energy metabolism in the cells. AMPK activation stimulates catabolic pathways that produce ATP and simultaneously inhibits ATP-consuming anabolic pathways, thus adjusting the cellular energy balance [START_REF] Hardie | The AMP-activated protein kinase pathway--new players upstream and downstream[END_REF][START_REF] Cheung | Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding[END_REF]. Viability, motility and acrosome reaction are important factors influencing fertilization quality. Better knowledge of signal transduction pathways regulating these indicators is important to improve in vitro fertilization. In mammals, very few studies show signal transduction pathways regulating sperm motility and acrosome reaction such as PKA, PKC, MAPK3/1 or PIK3 and MAPK14 [START_REF] Naz | Role of tyrosine phosphorylation in sperm capacitation/acrosome reaction[END_REF][START_REF] Almog | Identification of extracellular signal-regulated kinase 1/2 and p38 MAPK as regulators of human sperm motility and acrosome reaction and as predictors of poor spermatozoan quality[END_REF]. Moreover, a recent study has shown that an AMPK protein in boar spermatozoa was regulating sperm mobility [START_REF] Bragado | AMP-activated kinase AMPK is expressed in boar spermatozoa and regulates motility[END_REF]. In birds, Ashizawa et al. showed the presence of PKC in spermatozoa and its possible inhibitory effect on acrosome reaction [START_REF] Ashizawa | Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A[END_REF]. In 2009, Lemoine et al. showed that PKA and MAPK1 are positively involved in acrosome reaction and in the motility of chicken [START_REF] Lemoine | Potential involvement of several signaling pathways in initiation of the chicken acrosome reaction[END_REF]. In our study, we demonstrate that AMPK protein is present in chicken sperm and is involved in the regulation of chicken spermatozoa viability, mobility and acrosome reaction by using pharmacological activators (AICAR and Met) and inhibitor of AMPK (CC).

Materials and methods

1. Chemicals and reagents 6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo [1,5-a] pyrimidine (CC), AICA riboside (AICAR) and 1,1-dimethylbiguanide hydrochloride (Met) were obtained from Calbiochem. Stock solution of Met and AICAR were prepared in deionized water and stock solution of CC was prepared in dimethylsulfoxide (DMSO). PI/Sybr 14 (LIVE/DEAD sperm viability kit) was purchased from Molecular Probes.

Animals and semen collection

The animals used were 22-55 weeks-old adult Gallus domesticus. The semen was collected by the dorso-abdominal massage method [START_REF] Burrows | An Operation for the Correction of Madelung's Deformity and Similar Conditions: (Section of Orthopaedics)[END_REF]. After removing seminal plasma, sperm pellets were diluted in Beltsville Poultry semen extender (BPSE) [START_REF] Sexton | A new poultry semen extender. 1. Effects of extension on the fertility of chicken semen[END_REF] to a final sperm concentration of 500.10 6 cells/ml. Sperm samples were then incubated at 35°C for 25 min in presence or absence of CC or Met or AICAR. The corresponding controls with the vehicles alone were measured in parallel.

Spermatozoa quality assessment

The spermatozoa viability was measured by Sybr 14 and PI (propidium iodide). The spermatozoa mobility parameters were evaluated by the computer-assisted sperm analysis (CASA) system with an HTM-IVOS (Hamilton-Thorn Motility Analyzer, IVOS) [START_REF] Blesbois | Predictors of success of semen cryopreservation in chickens[END_REF] and the accomplishment of acrosome reaction was detected by FITC-conjugated peanut agglutinin (FITC-PNA) in presence of Ca 2+ and inner perivitelline layer (IPVL) [START_REF] Lemoine | Potential involvement of several signaling pathways in initiation of the chicken acrosome reaction[END_REF].

3. Immunocytochemistry Sperm cells were fixed in formaldehyde, and blocking with goat serum in PBS (Sigma). The sperm cells were then incubated overnight at 4°C with anti-phospho AMPKα (in PBS-goat serum) and then incubated with anti-rabbit IgG H+L (Southern biotech) (in PBS-goat serum). Sperm cells were incubated with Cy2-Streptavidin (Southern biotech) (in PBS) and incubated with DAPI (Sigma). Another experiment was carried out at same time in which sperm cells were incubated without anti-rabbit IgG and used as negative controls.

Statistical analysis

The data are expressed as mean ± S.E.M. Statistical analysis used SPSS v16.0. The significance of the difference between the treatments was calculated by Student t-test or analysis of variance (ANOVA). The level of significance was set at p<0.05.

Results

Localization of AMPK in chicken spermatozoa

The expression of AMPK protein in chicken spermatozoa was assessed with indirect immunofluorescence using anti-AMPKα as primary antibodies. Figure 1A, shows that AMPK protein is observed in the acrosome region, in the midpiece and in the entire flagellum. Rabbit IgG was used as a negative control and showed no positive signal in Figure 1C. 2A). AMPK localization (green) (Fig. 2B). Negative control with anti-rabbit IgG (Fig. 2C).

(B) (C) (A) (B)

Modulators effects on sperm viability

Before working with these concentrations, we assayed four different concentrations for each modulator and decided to keep the one giving the best results. Regarding the time factor, we chose to make our measurements after 25 min of incubation because AMPK expression on sperm quality is very clear at this time. The percentage of spermatozoa viability assessed using PI/SYBR14 was significantly increased under AICAR (by 80%, n=10) and under Met (by 79%, n=10) compared to control samples (Fig. 2A). Treatment with AMPK inhibitor CC did not affect spermatozoa viability.

Modulators effects on sperm mobility

Our results indicate that sperm motility was significantly increased in the presence of Met (~ 65%, n=10) and ~ 58% (n=10) under AICAR compared to the control sample. In contrast, incubation with CC significantly decreased the percentage of motile spermatozoa to ~ 33% (n=10) compared to the control (DMSO). Data are shown in Fig. 2B.

Modulators effects on sperm acrosome reaction

Discussion

AMPK was shown for the first time in mammalian tissues by Cheung et al. in 2000 [2]. It was also shown that it could be stimulated by uptake of glucose and oxidation of fatty acids [START_REF] Cheung | Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding[END_REF]. The present study demonstrated that AMPK protein is expressed in chicken spermatozoa lysates. The presence of AMPK proteins in the spermatozoa was confirmed by immunocytochemistry. AMPKα is present at high concentration in the flagellum and it also found in the acrosome and in the midpiece. In mammalian, AMPK proteins were shown in spermatozoa by Bragado et al. in 2012 [START_REF] Bragado | AMP-activated kinase AMPK is expressed in boar spermatozoa and regulates motility[END_REF], but none were found in the flagellum. We also showed that AMPK inhibitor (CC) negatively affects sperm motility and rate of acrosome reaction but not sperm viability. These results are in agreement with earlier studies by Bragado et al. [START_REF] Bragado | AMP-activated kinase AMPK is expressed in boar spermatozoa and regulates motility[END_REF], which showed that CC in chicken spermatozoa may reduce sperm motility but does not affect viability. Our results also suggest that AMPK activators (Met and AICAR) stimulate sperm viability, motility and rate of acrosome reaction.

In summary, we have shown that the AMPK pathway could be involved in the regulation of spermatozoa functions.

Figure 1 :

 1 Figure 1 : Immunolocalization of AMPK in chicken sperm. Images show the AMPK localization (green) and DAPI labelled nuclei (blue) (Fig.2A). AMPK localization (green) (Fig.2B). Negative control with anti-rabbit IgG (Fig.2C).

  Fig.2Cshows that the percentage of successful acrosome reaction was significantly increased by Met and AICAR after 25 min of incubation. Treatment of spermatozoa with CC significantly decreased the acrosome reaction rate: it is at ~ 15 % whereas it is at ~ 20 % in control (DMSO) sample.

Figure 2 :

 2 Figure 2 : Effect of AMPK modulators in the percentage of viable, motile spermatozoa and in the percentage of spermatozoa acrosome reaction. Spermatozoa were incubated with an AMPK modulators (in black) and with SHAM (in white). Experiment was performed 10 times; values (%) are mean ± SEM. a,b: significantly different ( P< 0.05)

Acknowledgements

We also thank F. Seigneurin, M. Magistrini for methodological discussions and the whole team that took care of the animals for helping me to collect the chicken semen.


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text:
The presence of iron pan limits rooting depth thus affecting available water content for plants and vulnerability of trees to storms. In some cases it may also limit the water infiltration rate and cause anaerobic conditions. The study was carried out in a region covering more than 1,150,200 ha in southwestern France: the region 'des Landes de Gascogne'. This region is mainly covered by sandy PODZOLS containing an iron pan, called 'alios', which is generally not continuous. Iron pan is geographically distributed as little patches in the landscape, which makes its mapping difficult. Moreover, constraints about accessibility strongly limit the number and the density of field observations that can be performed. Therefore, we tested the potential of a road-side survey sampling and subsequent digital soil mapping techniques to map the probability of iron pan occurrence at a regional level.

Field observations included 4,102 auger borings and 286 soil pits, corresponding to a mean sampling density of 0.38 observation per km 2 The sampling strategy corresponded mainly to a road-side survey. The presence/absence of an iron pan was registered.

The geographical coordinates of each sampling site were recorded with an average precision of + 5 m.

o • • • • • •
dppr on 3 × 3 (dppr3_3), 7x7 (dppr7_7), 21x21 (dppr21_21) and 41x41 (dppr41_41) pixel windows • o

MODELLING & VALIDATION STUDY AREA

We mapped this key diagnostic soil property as proposed by Kempen et al. (2012). In order to map the probability of the presence of an iron pan, we calibrated a SCORPAN regression kriging model which combines a logistic regression and a variogram analysis of the residuals. Using the GSIF R package, regression-kriging model was fitted by using the fit.gstatModel function. This function runs also a stepwise selection step. Next, the probability of "presence" of the iron pan was predicted at the nodes of a grid with 250-m resolution. The map was validated with a 5-fold cross validation.

The occurrence of iron pan was successfully mapped at a regional level. From a practical point of view this map could help regional planning for forestry and agriculture (e.g. choosing tree species for plantation, estimating irrigation needs of summer crop). Further work should be conducted including new potentially relevant co-variates and testing other modelling approaches to improve the prediction in this area. A next step will be the spatial prediction of the effective rooting depth as recommended by the GlobalSoilMap project (Arrouays et al., 2014).

DATA

The study area was located in southwestern France approximately between 43.3 and 44.5°N and 1.45°W to 0.10°E (Fig. 1). The 'Landes de Gascogne' is a nearly flat plain with elevation ranging from 0 to 256 m above sea level and slopes generally less than 0.5% (Fig. 2). The region receives about 900 mm year -1 of rain, and has a mean annual temperature of 12.7°C. The parent material of the soils is known as 'sables des Landes', which originated from tertiary fluvial alluviums that were spread by successive wind erosion periods during the Pleistocene. The soils that developed from this parent material are mainly PODZOLS. Some of these PODZOLS are characterized by the presence of a very hard iron pan, cemented by organic compounds and Al and Fe oxydes (Picture 1). The profile development is directly related to the seasonal variations of the water table. The depth and seasonal variations of the water table are mainly controlled by: i) the distance and relative height to small valleys, ii) an eolian micro-relief mainly consisting of a succession of low ridges and shallow troughs, ranging from about 0.30 to 1.50 m high and 10-50 m wide (Picture 2).

The co-variates related to DEM derivatives were found the most significant for explaining the presence of iron pan (Tab. 1).

VARIOGRAM

The variogram of the residuals exhibited a spatial structure as observed by the nugget and range (Fig. 5).

The fixed effect shows that iron pan occurrence is mainly located on rather flat areas, in interfluve position (Fig. 6). The map of the probability of iron pan occurrence (Fig. 7), shows large patches of iron pan that are not always corresponding to the fixed effect. The final map of the probability of iron pan occurrence clearly shows that the fixed effect alone is not sufficient to map this diagnostic soil property. It suggests that the co-variates used did not allow to explain all the controlling factors of the occurrence of iron pan. This may be attributed to the fact that the resolution of the DEM we used is not precise enough to capture very local variations in the micro-relief. Moreover, the nugget effect suggests that very local variations may be encountered. However, our results demonstrate that gathering numerous field informations helps to provide a relevant regional map when combining logistic regression and geotatistical analysis of the residuals. Conversely, these results suggest that our mapping excercice may suffer from limitation related to the spatial coverage of the dataset. Indeed, in the less accessible areas where less intensive sampling was conducted we may have missed some patterns of iron pan "presence" or "absence".

MAPPING

  Arrouays D., Grundy M. G., Hartemink A. E., Hempel J. W., Heuvelink G.B.M., Hong S.Y., Lagacherie P., Lelyk, G., McBratney A. B., McKenzie, N. J. Mendonça-Santos M.D., Minasny B., Montanarella L., Odeh, I. O. A., Sanchez P. A., Thompson J. A., Zhang G. L. 2014. GlobalSoilMap: towards a fine-resolution global grid of soil properties. Advances in Agronomy, 125, 93-134. Kempen, B.; Brus, D.J.; Vries, F. de; Engel, B. (2012) Updating legacy soil data for digital soil mapping. In Digital Soil Assement and beyond. Editors Minasny B., Malone B.P. & McBratney A.B. Taylor & Francis Group, 91-96 pp.

Estimate Std.Error z value Pr(>|z|)

	hppr	-6.785e-02 1.073e-02	-6.321	2.60e-10 ***
	slope	-1.272e-01 2.935e-02	-4.333	1.47e-05 ***
	elevation	-4.124e-03 1.138e-03	-3.624	0.00029 ***
	dppr41_41	1.308e-03 2.398e-04	5.455	4.89e-08 ***
	clc
	(Forests of deciduous	-1.115e+00 3.685e-01	-3.027	0.00247	**
	trees )
	dppr7_7	3.913e-04 1.690e-04	2.315	0.02059

* RELATIVE IMPORTANCE OF CO-VARIATES Signif. codes: 0 '***' 0.001 '**' 0.01 '*' INRA,

Unité InfoSol, US 1106, CS 40001 Ardon, Orléans cedex 2, France anne.richer-de-forges@orleans.inra.fr


Quentin Rougemont

Sophie Launey

Arnaud Gaigher

Anne-Laure Besnard

Emilien Lasne

Guillaume Evanno

Genome Wide Divergence Among population pairs of parasitic and non-parasitic lampreys (Lampetra fluviatilis and L. planeri)

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

hypothesis of eastern Atlantic affinities of salmon in southeastern Newfoundland This work highlights the utility of RAD-seq based approaches for the resolution of complex spatial patterns, further resolves a region of trans-Atlantic secondary contact in Atlantic salmon, and reinforces the difficulty in identifying loci associated with adaptive divergence under complex demographic scenarios T-205A-13 Detecting Genotypic Changes Associated with Selective Mortality at Sea in Atlantic Salmon: Polygenic Multilocus Analysis Surpasses Genome Scan Vincent Bourret*, Universite Laval, Melanie Dionne, Ministere du Developpement Durable, de l'Environnement, de la Faune et des Parcs and Louis Bernatchez, IBIS, Université Laval Here, we genotyped 5568 SNPs in Atlantic salmon populations across two cohorts to test for differential allelic and genotypic frequencies between juveniles (smolts) migrating to sea and adults (grilses) returning to freshwater after one year at sea Given the complexity of the traits potentially associated with sea mortality, we contrasted the outcomes of a single-locus F ST based genome scan method with a new multilocus framework to test for genetically-based differential mortality at sea While numerous outliers were identified by the single-locus analysis, no evidence for parallel, temporally repeated selection was found In contrast, the multi-locus approach detected repeated patterns of selection for a multi-locus group of 34 SNPs in one of the two populations No significant pattern of selective mortality was detected in the other population, suggesting different causes of mortality among populations Overall, these results support the hypothesis that selection mainly causes small changes in allele frequencies among many co-varying loci rather than a small number of changes in loci with large effects Consequently, moving away from the "selective sweep paradigm" and towards a multi-locus genetics framework may be a more useful approach for studying the genomic signatures of natural selection on complex traits in wild populations The degree of speciation between the European 'paired' species Lampetra planeri (Lp) and Lampetra fluviatilis (Lf) is unclear as these taxa are morphologically and phylogenetically very similar and may represent two ecotypes of a single species We investigated the level of gene flow between Lf and Lp populations in France using population genetics (13 microsatellites loci) and genomics (RAD sequencing) approaches We aimed at i) comparing the estimates of gene flow between sympatric Lf and Lp populations obtained with microsatellite and RAD markers and ii) identifying RAD loci putatively involved in the adaptive divergence between species Results based on the microsatellite genotyping of 665 individuals from six river systems revealed moderate to strong levels of gene flow between Lf and Lp populations sampled in the same rivers Estimates of genetic differentiation were much higher among Lp than Lf populations with the latter displaying higher levels of genetic diversity Overall, these results support the hypothesis that Lf and Lp are two ecotypes of a single species but common garden experiments are required to validate this hypothesis These results also suggest that the connectivity between Lp and Lf populations should be restored to maintain high levels of genetic diversity in Lp populations

  T-205A-14 Genome-Wide Divergence Among Population Pairs of Parasitic and Non-Parasitic Lampreys (Lampetra fluviatilis and L planeri) Quentin Rougemont*, INRA UMR Ecologie et Santé des Ecosystèmes, Sophie Launey, INRA UMR Ecologie et Santé des Ecosystèmes, Arnaud Gaigher, Université de Lausanne, Anne-Laure Besnard, INRA UMR Ecologie et Santé des Ecosystèmes, Emilien Lasne, INRA UMR CARRTEL and Guillaume Evanno, INRA UMR Ecologie et Santé des Ecosystèmes


text:
Vera Leatitia Mulder

Marine Lacoste

Nicolas N Saby

Dominique D Arrouays

Large-extent digital

published or not. The documents may come

General information

Total soil depth (SD t ) plays a key role in supporting various ecosystem services and properties, including plant growth, water availability and carbon stocks. Therefore, predictive mapping of SD t has been included as one of the deliverables within the GlobalSoilMap (GSM) project. In this work SD t was predicted for France using 2 different methods 1) Data mining, including a bias correction + kriging of residuals (DM) and 2) Multi-Resolution Kriging for large datasets (MrK).

Methods Data

The SD t was determined for 2116 sites, originating from the French Soil Monitoring network (RMQS). This dataset encompasses a broad spectrum of climatic, soil and agricultural parameters and covers the entire metropolitan France, based on a regular, 16km x 16km grid. Soil depth ranged from 0 to 300 cm, with a mean value of 102 cm.

Results

Large-extent digital soil mapping approaches for total soil depth

Outlook

Data mining MrK

Sample locations

  Deterministic trend: limited to a linear model Straight forward modelling approach Flexible in delivering spatial explicit uncertainty measures Potential for modelling beyond the country level, at high resolution Prediction of extremes values  (bias correction) Deterministic trend: flexible model choice, flexible for large datasets, high resolution Estimation of uncertainties: no direct of and not flexible for large datasets Promising prediction

INRA UR 0272 Science du Sol, Orléans, France Predicted soil depth Spatial uncertainties Environmental data Soil samples

	Topography	Vegetation	Soil/Geology
	Elevation	Land use	Parent material
	Slope	Forest type	Bare rock areas
	Aspect CTI Roughness	Climate Precipitation	Soil waterlogging indices
	Exposition	Temperature
	Curvature	Climate type
	Scale position

V.L. Mulder, M. Lacoste, N.P.A. Saby & D. Arrouays INRA US Infosol &

Bias correction Major findings DM + Kriging of residuals Multi -resolution kriging Deterministic trend Interpolation residuals Prediction uncertainty

	Data mining (R package				Calculation of prediction
	GBM)			Ordinary kriging		intervals by ordinary
	Bias correction					kriging of error
				Multi-resolution kriging		Calculation of
	Linear regression model	using spatial basis function for estimating		confidence intervals by Gaussian simulation of
				the covariance matrix		the kriging model
	Data mining		Difference		MrK
	Min	Q1	Mean	Median	Q3	Max	sd
	Data Mining	0	66	99	97	125	288	45
	MrK	4	78	95	96	112	193	25

Data mining: prediction interval MrK: confidence interval

									Min	Q1	Mean	Median	Q3	Max	sd
								Data Mining	0	97	111	113	127	197	25
								MrK	13	35	38	38	42	72	6
	Before correction After correction			Empirical adjustment (bias correction)of
				the distribution of variables using quantile
	Predicteded soil depth (cm)			mapping" -1) Identifying the distribution parameters of a population (RMQS data) 2 steps correction method:	 No extremes values
				-2) Apply these parameters to the
					predictive data to correct its distribution
	Observed soil depth (cm)
		Min	Q1	Mean	Median	Q3	Max	sd
	Observed	0	60	102	100	130	300	52
	Before	1	79	96	97	113	178	27
	After	0	68	102	98	130	269	49

R²: 0.55, 0.55 Lin's coef: 0.60, 0.75


text:
Yves Brunet

Amandine Gales

Jean-Jacques Godon

Nathalie Wéry

Estimating the bacterial source strength and the downwind influence of outdoor composting platforms

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

Oral 2484

Estimating the bacterial source strength and the downwind influence of outdoor composting platforms Y. Brunet 1 , A. Galès 2 , J.-J. Godon 2 , N. Wéry 2 1 ISPA, INRA, 33140, Villenave d'Ornon, France; 2 LBE, INRA, 11100, Narbonne, France Active and passive emission processes at composting sites result in the release of microorganisms that can then be transported by the atmospheric flow over some distance before they settle. Determining the impact of compost operations on bacterial source strength and on the distance from the site at which air concentration returns to its background value (the 'return distance') is a topic of importance for risk assessment. To this purpose, experimental campaigns were conducted in France at two outdoor composting sites, where air concentration measurements were performed at several locations downwind from the source, along horizontal transects aligned with the mean wind direction. All relevant microclimatic variables were also registered. Quantitative PCR was used to monitor the microbial indicators selected in a previous study in order to trace specifically the bioaerosols emitted from compost piles and evaluate their dynamics in the atmosphere. A simple Gaussian-type modelling approach was designed to estimate the source strength and the return distance from the measured concentration transects. The main factors of variation in terms of air concentration, source strength and return distance were found to be the site itself, then the operation type: overturning generates the largest amount of bacteria, followed by loading and screening. This affects the return distance, which also shows a weak variation with mean wind velocity. The generic character of this method combining atmospheric transport modelling with air concentration measurements makes it applicable to other cases of atmospheric dispersal from point or area sources.

Keywords: Compost, Atmospheric dispersal, Microbial aerosol.


Albert Ribas-Agusti

Barbara Gouble
email: barbara.gouble@avignon.inra.fr

Jean-Marc Marc Audergon

Mohamed El Maataoui

Catherine M G C Renard

Mohamed El Maataoui

Albert Ribas-Agustí

Goldrich Cooked Goldrich

Raw Orangered

Microstructural changes in cooked apricots related to the loss of texture

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

Variety largely influenced the loss of texture (Fig. 1). Orangered was the variety with higher resistance to the texture loss (67% of the initial firmness), followed by Hargrand (74%), Iranien (75%) and Goldrich (86%). The tissues did not show severe damaging after cooking, however, some microstructural changes could be observed. The cells lost turgor as demonstrated by the higher sinuosity of the cell walls. Some intercellular spaces swelled, creating round-shaped microstructures filled with unmethylated pectin, as showed by LM19 (Fig. 2). Their presence across the varieties showed to be in line with their resistance to the loss of texture (Fig. 3). Varietal differences in the biochemical properties must have prompted the ease of pectin to be leached from the tissue or to be retained within the intercellular spaces.

RESULTS & DISCUSSION

INTRODUCTION

Fruits were selected from four varieties at similar ripening stages, which were determined by their firmness using a compression test [1]. The apricot halves were cooked at 85 °C in sugar syrup and their texture was measured by the maximum force in the penetrometry test [1]. Microstructure changes were revealed by ruthenium red staining (pectin) and LM19 immunolabelling (unmethylated and low-methylated pectin) [2].

METHODOLOGY

We identified a link between the creation of intercellular microstructures and the loss of texture. This study opens the possibility of understanding the creation of these microstructures and the biochemical properties that are implied, which is in the interest of the food industry for the elaboration of higher quality apricot products.

CONCLUSIONS

  et Qualité des Produits d'Origine Végétale, INRA, Avignon, France b UMR408 Sécurité et Qualité des Produits d'Origine Végétale, Université d'Avignon, Avignon, France c UR1062 Unité de Génétique et d'Amélioration des Fruits et Légumes, INRA, Avignon, France d EA 4279 Laboratoire Physiologie des Fruits et Légumes, Université d'Avignon, Avignon, France

Fig. 1 .Fig. 3 .

 13 Fig. 1. Loss of texture after thermal treatment as expressed by the percentage loss of the initial firmness (Fmax, penetrometry test). The boxplots indicate means (red crosses), medians (central horizontal bars), first and third quartiles (lower and upper limits respectively), and minimum and maximum values (blue points).

Fig. 2 .

 2 Fig. 2. Detail of the round intercellular spaces filled with unmethylated pectin in a thermally treated sample (Iranien). A, ruthenium red staining; B, LM19 immunolabelling.

The quality of the apricot products is largely defined by their texture, which is significantly lost upon processing. The pectin properties of apricots may play an important role in this loss of texture, even more than the initial firmness, but this is not yet well understood[1]. This study aims to identify a varietal differentiation in the texture-loss properties and to link them with microstructural changes, with a final goal of understanding the mechanisms that influence the texture loss in apricot.

This work was financed by the Agence Nationale de la Recherche under the project ILLIAD (ANR-11-ALID-0006) The authors gratefully acknowledge UPPIA and the Prix de Recherche en Nutrition Institut Appert 2014 for economic support