Jorge Diogène
email: jorge.diogene@irta.cat

Laia Reverté

Maria Rambla-Alegre

Vanessa Del Río

Pablo De La Iglesia

Mònica Camp

Oscar Palacios

Cintia Flores

Josep Caixach

Christian Ralijaona

Iony Razanajatovo

Agathe Pirog

Hélène Magalon

Nathalie Arnich

Jean Turquet

Identification of ciguatoxins in a shark involved in a fatal food poisoning in the Indian Ocean

Severe food poisoning events after the consumption of sharks have been reported since the 1940s; however, there has been no clear understanding of their cause. Herein, we report for the first time the presence of ciguatoxins (CTXs) in sharks. The identification by mass spectrometry of CTXs, including two new analogues, in a bull shark (Carcharhinus leucas) that was consumed by humans, causing the poisoning and death of 11 people in Madagascar in 2013 is described. Typical neurotoxic ciguatera symptoms were recorded in patients, and toxicological assays on extracts of the shark demonstrated CTX-like activity. These results confirm this episode as a ciguatera poisoning event and expand the range of pelagic fish species that are involved in ciguatera in the Indian Ocean. Additionally, gambieric acid D, a molecule originally described in CTX-producing microalgae, was identified for the first time in fish. This finding can contribute to a better understanding of trophic relations within food webs. The present work confirms that consumption of sharks from the Indian Ocean should be considered a ciguatera risk, and actions should be taken to evaluate its magnitude and risk in order to manage shark fisheries.

Ciguatera is a well-known food poisoning that occurs when fish containing ciguatoxins (CTXs) are consumed. These potent neurotoxins are produced by microalgae of the genus Gambierdiscus 1-3 and Fukuyoa 4, 5 . Ciguatoxins produced by these microalgae may be transferred along the food web, eventually reaching carnivorous fish like barracuda or amberjack. Ciguatera is the main cause of seafood poisoning due to the consumption of fish, and estimations point out around 50,000-500,000 people are affected by ciguatera each year 6 , although these should be re-evaluated for a better assessment of the present impact of ciguatera.

 . In this particular event, patients presented almost exclusively neurological symptoms. Boisier et al. identified two toxic extracts from the liver of the shark, which were proposed to be the causative agent of the poisoning, and tentatively named the new toxins as carchatoxin-A and carchatoxin-B 9 . However, the toxicity levels of the shark flesh did not match that of the liver extracts, and thus, toxicity remained unexplained. No further information

regarding the chemical behaviour, structure, toxicity or mechanism of action of carchatoxins has been published since then. Another event, occurring between November 14 and 19, 2013 at Fenerive-Est in Madagascar, caused the poisoning of 97 people that presented ciguatera symptoms after eating the flesh, the liver or the head of a bull shark, 11 of whom died. Our preliminary laboratory results of this particular event were communicated to the French "Agence nationale de sécurité sanitaire de l'alimentation, de l' environnement et du travail" (ANSES) in order to quickly manage the potential risk of food poisoning by shark consumption in the Madagascar area [START_REF]par des ciguatoxines: occurrence, méthodes analytiques, cas humains rapportés et éléments d' éthologie[END_REF] . Further data regarding the epidemiology of this event, described that the major symptoms were neurological and digestive [START_REF] Rabenjarison | Intoxication à la ciguatoxine après consommation de requin à Fenerive-Est: profil épidémio-clinique et résultats de laboratoire. Ciguatera poisoning after consumption of shark in Fenerive-Est: epidemiological and clinical aspects and laboratory results[END_REF] .

We report herein the confirmation of ciguatera, caused by consumption of this bull shark (C. leucas) in Madagascar in November 2013. This was based on the evidence of symptoms in patients and in mice, cellular toxicity, and unequivocal identification of CTXs by liquid chromatography coupled to high resolution mass spectrometry (LC-ESI-HRMS). To the best of our knowledge, this is the first identification of CTXs in sharks.

Results

Poisoning event in Madagascar. In November 2013, an outbreak of fish poisoning following the consumption of shark was reported in the district of Fenoarivo Atsinanana (Fenerive-Est, Madagascar). According to the information transmitted to the ANSES by the "French Institute for Public Health Surveillance, " dated 22 April 2014, 124 people, 11 of whom died, were poisoned after consuming the flesh, liver, head and part of the viscera of a shark [START_REF] Rabenjarison | Intoxication à la ciguatoxine après consommation de requin à Fenerive-Est: profil épidémio-clinique et résultats de laboratoire. Ciguatera poisoning after consumption of shark in Fenerive-Est: epidemiological and clinical aspects and laboratory results[END_REF] . The patients developed symptoms between 2 and 12 h following ingestion of their meal, and the predominant neurological signs were paraesthesia of the extremities, dysesthesia, and reversing sensitivity of hot and cold. These symptoms were accompanied by headache, dizziness, and arthralgia. The digestive symptoms were moderate and inconsistent. The clinical profile was similar to that of patients that had previously been poisoned after consumption of shark in Madagascar [START_REF] Boisier | Fatal mass poisoning in madagascar following ingestion of a shark (Carcharhinus-leucas) -Clinical and epidemiologic aspects and isolation of toxins[END_REF] . A detailed epidemiological report is presented by Rabenjarison et al. [START_REF] Rabenjarison | Intoxication à la ciguatoxine après consommation de requin à Fenerive-Est: profil épidémio-clinique et résultats de laboratoire. Ciguatera poisoning after consumption of shark in Fenerive-Est: epidemiological and clinical aspects and laboratory results[END_REF] , and additional investigations conducted by agents of the "Institut Halieutique et des Sciences Marines" (Tulear, Madagascar) concluded that the shark in question was a female of about 1.5 m in length. Samples of the fish implicated in the episode, and used in our study, consisted of salted stomach, three dried fins, and partially cooked flesh. The genetic analyses that we performed using 22 microsatellite loci on the five samples demonstrated that they all belong to the species C. leucas and surely to the same individual (identical multilocus genotypes; Supplementary Table S1 and Fig. 1).

Toxicity evaluation of shark samples by mouse bioassay (MBA).

The MBA performed on the flesh samples showed toxicity, although quantification was not possible due to the limited amount of samples. The single dose injected corresponds to an amount of 50 g equiv. flesh/mouse. The symptoms observed in the mice included: paralysis of limbs, dyspnoea, convulsions, mild diarrhea, and mortality from respiratory failure between 3 and 4 hours after injection of the extract. Toxicity and symptoms in mice were similar to those previously described in the study on shark toxins in Madagascar [START_REF] Boisier | Fatal mass poisoning in madagascar following ingestion of a shark (Carcharhinus-leucas) -Clinical and epidemiologic aspects and isolation of toxins[END_REF] . The MBA performed on the stomach sample showed a very high toxicity. Even at low doses, when mortalities were observed these were always rapid (survival time less than 1.5 h) and otherwise the mice recovered. The symptoms observed were dominated by neurological problems, including difficulty with breathing, followed by a severe respiratory arrest. In some mice, hyper-salivation was also observed. The lowest dose tested that resulted in the death of mice was estimated at 72 mg equiv. stomach per mouse of 20-22 g. Fin samples were not tested by MBA.

Evaluation of ciguatoxin-like activity by Neuro-2a cell-based assay (CBA).

Neuro-2a cells exposed to Pacific ciguatoxin P-CTX-1 (CTX1B) standard presented the expected dose-response curve with a regression factor (R 2 ) of 0.996. The concentration of P-CTX-1 that caused a 50% cell mortality (IC 50 ) was 19.9 pg/mL. The limit of detection (LOD), defined as the concentration of P-CTX-1 that causes a 20% of cell mortality (IC 20 ), was 8.4 pg/mL (Supplementary Figure S2). Considering a maximum exposure concentration of 200 mg/mL for flesh, 100 mg/mL for stomach and fins 1 and 3, and 50 mg/mL for fin 2 (Supplementary Figure S3), the effective LODs (eLODs) for P-CTX-1 in shark samples were 0.04 µg P-CTX-1 equiv./kg for flesh, 0.08 µg P-CTX-1 equiv./kg for stomach and fins 1 and 3, and 0.17 µg P-CTX-1 equiv./kg for fin 2. Flesh, stomach and fins 1 to 3 crude extracts of C. leucas contained 0.06, 92.78, 0.12, 0.79 and 0.17 µg P-CTX-1 equiv./kg matrix, respectively (Table 1).

With the aim of separating the different compounds to better identify the toxin profile of the most toxic sample, the stomach extract was fractionated by high-performance liquid chromatography (HPLC). In order to identify the distribution of the toxin within the 28 fractions recovered, cells were first exposed to 2.17 µL of each fraction/mL. CTX-like activity was observed in fractions F8 to F22. Fractions F8 and F17 to F22 fell within the working range (IC 20 -IC 80 ), and CTX-like content was able to be quantified. The use of lower fraction volume (0.27 µL of each fraction/mL) was required to quantify fractions F13 and F14 (Fig. 1). Further dilution was required to quantify fractions F9 to F12. Distribution of ciguatoxins after fractionation of the stomach crude extract is shown in Table 2.

Confirmation of ciguatoxins (CTXs) by liquid chromatography coupled to high resolution mass spectrometry (LC-ESI-HRMS).

A liquid chromatography electrospray ionization high-resolution mass spectrometry (LC-ESI-HRMS) method was developed for the analysis of CTXs in extracts of C. leucas, based on previous LC-MS/MS methods [START_REF] Caillaud | Monitoring of dissolved ciguatoxin and maitotoxin using solid-phase adsorption toxin tracking devices: Application to Gambierdiscus pacificus in culture[END_REF][START_REF] Yogi | Detailed LC-MS/MS analysis of ciguatoxins revealing distinct regional and species characteristics in fish and causative alga from the Pacific[END_REF][START_REF] Hamilton | Multiple ciguatoxins present in Indian Ocean reef fish[END_REF] . The spectra of I-CTXs were dominated by

[M+H] + , [M+NH 4 ] + , [M+Na] + , [M+H-H 2 O] + , [M+H-2H 2 O] + in
accordance with Hamilton et al. [START_REF] Hamilton | Multiple ciguatoxins present in Indian Ocean reef fish[END_REF] . The adduct ions giving higher signals, specifically [M+NH 4 ] + and [M+Na] + , were chosen for confirmation and quantification purposes.

Crude extracts of flesh, stomach and fin 2 were analyzed by LC-ESI-HRMS. The presence of CTX analogues was not observed in the flesh nor in the fin 2 crude extracts. A possible explanation is that the LOD attained by CBA (0.04 µg P-CTX-1 equiv./kg flesh tissue and 0.17 µg P-CTX-1 equiv./kg fin 2 tissue) is lower than the LOD attained by LC-ESI-HRMS (0.5 µg P-CTX-1 equiv./kg tissue). In the stomach crude extract, the CTX analogues I-CTX-1&2 and I-CTX-3&4 were detected and quantified (Table 1 From a qualitative point of view, both LC-ESI-HRMS and CBA showed the presence of P-CTX-1 equiv. in the stomach crude extract. However, lower contents of P-CTX-1 equiv. were estimated by LC-ESI-HRMS in relation to the CBA. This difference in the quantification could be attributed to the different principles of the techniques: while LC-ESI-HRMS is based on structural identification of specific CTX analogues, and may neglect some non-described CTX analogues, CBA measures a composite toxicity, which is a global response indicative of the toxic effect of several CTX analogues on cells.

Having identified the toxic stomach fractions using the CBA, fractions F8 to F22 were analyzed by LC-ESI-HRMS for toxin identification. Fractionation of the stomach crude extract reduced matrix interferences and confirmed the presence of I-CTX-1&2 and/or I-CTX-3&4 in fractions F9 to F12, the most toxic ones by CBA (Table 2). Extracted ion chromatograms for I-CTX-1&2 and I-CTX-3&4 found in fraction F12 from stomach are shown in Fig. 2a andd, respectively. Full HRMS exact mass spectra of I-CTX-1&2 and I-CTX-3&4 (Fig. 2b ande, respectively) confirmed the presence of these toxins. The isotopic pattern of each signal was taken into consideration in assigning their molecular formula. In addition, these toxins showed a profile similar to P-CTX-1 according to [M+NH 4 ] + and [M+Na] + . As for the analysis of crude extracts, the percentages of P-CTX-1 equiv. in fractions F9 to F12 determined by LC-ESI-HRMS were higher than those obtained by CBA. Nevertheless, both techniques concluded that fractions F9 to F12 contained the highest CTX content among all fractions.

Two new CTX analogues (I-CTX-5 and I-CTX-6) were detected by LC-ESI-HRMS (Fig. 2c andf). The identification of these new CTX analogues related to I-CTXs in the stomach crude extract and fractions was given using the restrictive criteria to propose elemental formulae. Ion assignment indicated that the new CTX analogues had nearly the same molecular formula as I-CTX-1&2 and I-CTX-3&4 with only 2H less, which corresponds to the formation of a double bond. The ring double bond equivalents (RDBEs) for both unknown compounds was 16.5, which corresponds to the 15.5 RDBE value from CTX analogues (I-CTX-1&2 and I-CTX3&4). Analogues were confirmed according to their theoretical accurate m/z, measured m/z, and mass accuracy (ppm): i) I-CTX- 2c and f confirm the presence of these toxins. Extracted ion chromatograms (Fig. 2g) and HRMS exact mass spectra (Fig. 2h) confirm the presence of GA D by the m/z of most abundant ion peaks [M+NH 4 ] + . No GA D was identified in the stomach or in the fins.

Discussion

The methodology presented in this paper, which combines a multi-disciplinary approach focused on epidemiology, toxicology and instrumental analysis, has proved to be effective in the identification of CTXs in seafood and contributes to a better characterization of the present incidence of ciguatera. This is the first evidence of the presence of CTXs in sharks. The identification of CTXs in the shark responsible for a food poisoning event in Madagascar presented herein, along with additional observations, confirm the episode as a ciguatera event and the suspected implication of sharks in ciguatera [START_REF] Meyer | An investigation into ciguatoxin bioaccumulation in sharks[END_REF] . The evidences that support this confirmation are: i) the symptoms observed in patients matched those of ciguatera, ii) injection of flesh and stomach crude extracts to mice resulted in symptoms characteristic of CTXs, which were comparable to those reported for I-CTX in the bony fish Lutjanus sebae from the Indian Ocean [START_REF] Hamilton | Multiple ciguatoxins present in Indian Ocean reef fish[END_REF] , iii) neuroblastoma cells exposed to flesh, stomach and fin crude extracts showed the characteristic toxicity of CTXs through activation of voltage-gated sodium channels, and iv) LC-ESI-HRMS provided the identification of I-CTX-1&2 and I-CTX-3&4 in the stomach extracts. The stomach was extremely toxic with an estimation of 92.78 µg P-CTX-1 equiv./kg by cell-based assay (CBA), a concentration approximately 10,000 times the guidance level concentration of P-CTX-1 causing ciguatera in humans, established at 0.01 µg P-CTX-1/kg by the FDA [START_REF] Dickey | A public health perspective[END_REF] , and considered by EFSA [START_REF] Efsa | Panel on Contaminants in the Food Chain; Scientific Opinion on marine biotoxins in shellfish-Emerging toxins: Ciguatoxin group[END_REF] as the level expected not to exert effects in sensitive individuals. The estimation of the stomach levels by LC-ESI-HRMS was lower than determined by CBA, 16.28 P-CTX-1 equiv./kg; however, this level is well above the FDA guidance level. Flesh and fins presented toxicity with an estimation by CBA of 0.06 µg P-CTX-1 equiv./kg in the flesh and 0.12, 0.79 and 0.17 µg P-CTX-1 equiv./kg in fins 1 to 3, respectively, which are also above the FDA guidance level. Identifying CTXs in viscera is significant, since local food habits from the Indian Ocean include eating liver and viscera [START_REF] Rabenjarison | Intoxication à la ciguatoxine après consommation de requin à Fenerive-Est: profil épidémio-clinique et résultats de laboratoire. Ciguatera poisoning after consumption of shark in Fenerive-Est: epidemiological and clinical aspects and laboratory results[END_REF] , some of which are dried and salted. Since the liver of the shark was not available, the possible presence of carchatoxins previously described in other shark poisoning events [START_REF] Yasumoto | Fish poisoning due to toxins of microalgal origins in the Pacific[END_REF] could not be studied.

Currently, only four I-CTX analogues (I-CTX-1&2 and I-CTX-3&4) have been described in the literature [START_REF] Hamilton | Multiple ciguatoxins present in Indian Ocean reef fish[END_REF] . As for the already known CTXs, I-CTX-1&2 and I-CTX -3&4, our results obtained in the stomach of shark revealed higher amount of I-CTX-3&4 in relation to I-CTX-1&2 (60% vs 40% of the total amount of I-CTXs estimated by LC-ESI-HRMS). Contrarily, in that previous study on Indian CTXs in one fish (Lutjanus sebae), a lower amount of I-CTX-3&4 in relation to I-CTX-1&2 was described [START_REF] Hamilton | Multiple ciguatoxins present in Indian Ocean reef fish[END_REF] . This difference may be explained by the tissue evaluated, since in their work the whole fish was analysed [START_REF] Hamilton | Multiple ciguatoxins present in Indian Ocean reef fish[END_REF] . Additionally, Hamilton and collaborators postulated that I-CTXs-1&2 might originate from dinoflagellates and that I-CTX-3&4 would be metabolites produced in fish [START_REF] Hamilton | Multiple ciguatoxins present in Indian Ocean reef fish[END_REF] . Since sharks are higher in the trophic webs than L. sebae, this may explain the higher amounts of I-CTX3&4 in relation to I-CTX-1&2 obtained in shark stomach. Herein, two new I-CTX analogues have been identified by LC-ESI-HRMS in the stomach extract and fractions, I-CTX-5 showing 2H less than I-CTX-1&2 and I-CTX-6 showing 2H less than I-CTX-3&4, which corresponds to the formation of a double bond. This result widens the number of CTXs analogues possibly present in nature [START_REF] Caillaud | Monitoring of dissolved ciguatoxin and maitotoxin using solid-phase adsorption toxin tracking devices: Application to Gambierdiscus pacificus in culture[END_REF][START_REF] Yogi | Detailed LC-MS/MS analysis of ciguatoxins revealing distinct regional and species characteristics in fish and causative alga from the Pacific[END_REF][START_REF] Hamilton | Multiple ciguatoxins present in Indian Ocean reef fish[END_REF] and this will impact our understanding of ciguatera. First, the identification of new CTX analogues may indicate that the metabolism of CTXs may be more complex than previously foreseen. Second, these new CTX analogues possibly present in seafood will need to be taken into account for a better evaluation of ciguatera risks.

Gambieric acid D (GA D) [START_REF] Fuwa | Total Synthesis and Complete Stereostructure of Gambieric Acid A[END_REF] was identified in shark flesh. To the best of our knowledge, this work is the first report of GA in any organism other than Gambierdiscus spp. [START_REF] Yasumoto | Fish poisoning due to toxins of microalgal origins in the Pacific[END_REF] . The identification of GA D in the flesh of the shark evidences, additionally to the presence of CTXs, the link between Gambierdiscus spp. and this particular food poisoning event. Identifying GA D in sharks that, as carnivorous pelagic fish are situated at the highest levels of the marine food webs, demonstrates how stable these molecules may be throughout their transfer and metabolic transformations along the food webs. The identification of molecules produced by microorganisms in animals situated at higher trophic levels, such as GA D would constitute, as for the analysis of fatty acids or stable isotopes, a good strategy to understand trophic relations in the ecosystems. Gambieric acid A has demonstrated no toxicity in mice, while a mixture of GA C and GA D was moderately toxic to mouse lymphoma cells L5178Y [START_REF] Nagai | Gambieric acids, new potent antifungal substances with unprecedented polyether structures from a marine dinoflagellate Gambierdiscus toxicus[END_REF][START_REF] Nagai | Gambieric acids -unprecedent potent antifungal substances isolated from cultures of a marine dinoflagellate Gambierdiscus toxicus[END_REF] . Additionally, GA A has been demonstrated to bind to the voltage gated sodium channels in synaptosomes isolated from rat brains [START_REF] Inoue | Inhibition of brevetoxin binding to the voltage-gated sodium channel by gambierol and gambieric acid-A[END_REF] in the same manner as CTXs, but with much less affinity to these than CTXs, possibly explaining its low toxicity. To better understand the potential harmful effects of GAs, and more specifically of GA D, their toxicity should be further characterized; however, at this moment reference material for GAs is not commercially available. Urgent need exists for the availability of certified standards for CTXs and GAs.

About 100 million sharks are caught each year world-wide [START_REF] Worm | Global catches, exploitation rates, and rebuilding options for sharks[END_REF] , and the global shark fin trade is estimated to be worth US $ 400-500 million a year [START_REF] Ioc) | A preliminary value chain analysis of shark fisheries in Madagascar IOTC-2015-WPEB-11-17[END_REF] . The identification of CTXs in shark from the Indian Ocean may favour the re-consideration of local food safety measures that could affect the shark fisheries industry, which is of special relevance in areas such Madagascar [START_REF] Ioc) | A preliminary value chain analysis of shark fisheries in Madagascar IOTC-2015-WPEB-11-17[END_REF] . The present work confirms that shark consumption, in this example, a bull shark (C. leucas), from the Indian Ocean should be considered a ciguatera risk, and actions should be taken to evaluate its magnitude and risk in order to manage shark fisheries. As for the numerous suspicious cases of ciguatera involving sharks [START_REF] Meyer | An investigation into ciguatoxin bioaccumulation in sharks[END_REF] , it may be postulated that sharks with CTXs will not be restricted to the species C. leucas and to the Indian Ocean. Consequently, other species of shark and other oceans should also be considered for ciguatera evaluation, especially to account for the migration of sharks and current changes in the geographical distribution of sharks due to fishing pressure and global warming [START_REF] Sguotti | Distribution of skates and sharks in the North Sea: 112 years of change[END_REF][START_REF] Lezama-Ochoa | Present and future potential habitat distribution of Carcharhinus falciformis and Canthidermis maculata by-catch species in the tropical tuna purse-seine fishery under climate change[END_REF] .

Methods

Samples. Shark samples were collected and analyzed in the framework of a research and development agreement funded by ANSES. Five samples were recovered by the health authorities of Madagascar "Agence de Contrôle Sanitaire et de la Qualité des Denrées Alimentaires de Madagascar" (ACSQDA) and were transferred to the laboratory by the WHO and the "Pasteur Institute of Madagascar". The salted stomach, three dried fins and partially cooked flesh samples were stored at -25 °C until extraction. Samples of flesh and stomach were crushed and homogenized before starting the analyses. Until extraction, they were stored at -25 °C.

Shark species identification through microsatellite genotyping and Dloop sequencing. The five samples (flesh, stomach and fins 1 to 3) from elasmobranch tissues, suspected to be from bull shark (C. leucas), were used for identification. Total genomic DNA was extracted using Qiagen DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany). The mitochondrial control region (D-loop; 832 bp) was also sequenced using primers designed previously [START_REF] Tillett | Evidence for reproductive philopatry in the bull shark Carcharhinus leucas[END_REF] , but failed to amplify due to DNA degradation before collection. Therefore, genotyping was performed using a total of 22 microsatellite loci [20 loci developed for C. leucas [START_REF] Pirog | Isolation and characterization of 20 microsatellite markers from Carcharhinus leucas (bull shark) and cross-amplification in Galeocerdo cuvier (tiger shark), Carcharhinus obscurus (dusky shark) and Carcharhinus plumbeus (sandbar shark)[END_REF] , one locus (Cli106) for C. Limbatus [START_REF] Keeney | Characterization of microsatellite loci isolated from the blacktip shark and their utility in requiem and hammerhead sharks[END_REF] and one locus (Gc01) for Galeocerdo cuvier [START_REF] Pirog | Isolation and characterization of eight microsatellite loci from Galeocerdo cuvier (tiger shark) and cross-amplification in Carcharhinus leucas, Carcharhinus brevipinna, Carcharhinus plumbeus and Sphyrna lewini[END_REF] ]. All amplifications were performed as described in the literature [START_REF] Pirog | Isolation and characterization of 20 microsatellite markers from Carcharhinus leucas (bull shark) and cross-amplification in Galeocerdo cuvier (tiger shark), Carcharhinus obscurus (dusky shark) and Carcharhinus plumbeus (sandbar shark)[END_REF] . Then, identical multi-locus genotypes (MLGs) were identified using the software GenClone v. 2.0 [START_REF] Arnaud-Haond | GENCLONE: a computer program to analyse genotypic data, test for clonality and describe spatial clonal organization[END_REF] and, using the software STRUCTURE v. 2.3.1 [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF] , assignment tests were performed together with MLGs obtained from other carcharhinid shark species (C. leucas, C. obscurus and C. plumbeus) that are known to amplify with the same loci.

Toxin standards. Pacific type 1 CTX (P-CTX-1 or P-CTX-1), P-CTX-2 and P-CTX-3 standard solutions were provided by Pr. Richard J. Lewis (The Queensland University, Australia). P-CTX-1 standard was used for CBA and LC-ESI-HRMS analysis. P-CTX-2, P-CTX-3 standards were used only for LC-ESI-HRMS analysis.

Flesh, stomach and fins extraction. Samples were extracted and purified according to the protocol described in the literature [START_REF] Lewis | In Manual on Harmful Marine Microalgae[END_REF] with minor modifications provided by ANSES. In brief, 10 g ± 0.1 g of flesh, stomach or fins homogenates were placed in 50-mL tubes. Samples were extracted in 20 mL of acetone and homogenized with an Ultraturrax blender. Samples were heated in the sealed tube at 70 °C for 10 min in a water bath. The supernatant was recovered by centrifugation at 3,000 g for 10 min at 4 °C and filtered using 0.45-µm nylon filters. The sample pellets were re-extracted with acetone and supernatants were pooled and evaporated until dry. Liquid/ liquid partition was then performed twice in the tubes with 20 mL of water/diethyl ether (DEE) (1:4, v-v). The DEE upper phase was recovered and pooled with the second DEE partition. Both DEE phases were evaporated until dry. The dried extracts were then dissolved in 4 mL of n-hexane and 2 mL of methanol/water (4:1, v-v). The hexane upper phase was removed. This liquid/liquid partition was repeated three times and the methanol phases were pooled and evaporated until dry. Finally, the resulting residues were re-dissolved in 4 mL of HPLC-grade methanol and preserved at -20 °C until analyzed.

Stomach extract fractionation.

A total of 2,750 µL of stomach extract were evaporated until dry using N 2(g) , and re-dissolved in 1,000 µL of HPLC-grade methanol. The analytical fractionation of this extract was performed as described before for the chromatographic separation of CTXs 3 . Once the chromatographic run started, fractions were collected every 30 seconds (n = 28). After fractionation, the volume of each tube was evaporated to dryness, re-dissolved in 500 µL of HPLC-grade methanol and preserved at -20 °C until analyzed. Stomach fractions were analyzed by LC-ESI-HRMS and CBA, but not by MBA.

Mouse bioassay.

The protocol used at HYDROREUNION was validated beforehand by the ethics committee (Protocol agreement n° EU0450 -GIP CYROI -APAFiS -Autor. APAFiS #2641-2015110916009490) and was in accordance with the regulations in force. This protocol is based on a standard method developed by ANSES (CATNAT-10). The extracts of shark were solubilized in Tween-60 1-5% saline solution, and then injected into three mice (male, OF1; 20 ± 2 g) by intraperitoneal (i.p.) route. The mice were observed continuously during the first 2 h, and then monitored regularly up to 24 h after injection. The interpretation of the results was based on the symptoms observed and the time-to-death of the mice. The typical symptoms of the presence of CTXs include profuse diarrhea, piloerection, respiratory disorders, dyspnoea and, when using male mice, transient pre-erectional cyanosis of the penis (which can become priapism). With the Indian Ocean toxins, this last symptom is observed only very rarely. It therefore does not appear in the classical description for this region. The death of 1 or 2 mice within 24 h was deemed a positive result indicating the presence of CTXs (sample therefore non-edible).

Assessment of ciguatoxin-like activity by Neuro-2a cell-based assay. Neuroblastoma mice

(Neuro-2a cell line: CCL-131) were purchased from the American Type Culture Collection (ATCC) (LGC standards S.L.U., Barcelona, Spain). The presence of CTX-like activity in shark tissues extracts was evaluated on Neuro-2a cells according to the method based on the use of ouabain and veratridine published by Caillaud and co-workers [START_REF] Caillaud | Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands[END_REF] . Briefly, cells were exposed to the P-CTX-1 standard and to shark extracts for 24 h, and the CTX-like activity was measured in the presence of ouabain and veratridine with the MTT colorimetric assay [START_REF] Manger | Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitive sodium channels: semiautomated assay for saxitoxins, brevetoxins, and ciguatoxins[END_REF] . Previous to the analysis of flesh, stomach and fins crude extracts or stomach fractions by CBA, methanol was removed from the extracts/fractions and P-CTX-1 standard solution by evaporation under N 2(g) and re-dissolved in RPMI medium. CTX-like activity was estimated with respect to P-CTX-1.

Liquid chromatography coupled to high resolution mass spectrometry (LC-ESI-HRMS). An

Orbitrap-Exactive HCD (Thermo Fisher Scientific, Bremen, Germany) mass spectrometer equipped with heated electrospray source (H-ESI II), a Surveyor MS Plus pump and an Accela Open AS auto-sampler kept isothermal at 15 °C (Thermo Fisher Scientific, San Jose, California) were used for the analysis by LC-ESI-HRMS.

The chromatographic separation was performed on a reversed-phase Hypersil Gold C 18 (50 mm × 2.1 mm, 1.9 µm) (Thermo Fisher, Scientific, Bremen, Germany) at a flow rate of 250 µL/min. Mobile phase A was water and B was acetonitrile/water (95:5), both containing 2 mM ammonium formate and 0.1% formic acid. The gradient elution program for the analysis was: 30% B 1 min, 30-40% B 2 min, 40-50% B 1 min, 50-90% B 5 min, 90% B 3 min and return to initial conditions for re-equilibrate (11 min 30% B). A 5-µL injection volume was used. The total duration of the method was 25 min.

The analyses were carried out in positive electrospray ionization (ESI+) mode, and the instrument was calibrated daily. P-CTX-1 was used to optimize the source, transmission and HRMS conditions in positive mode. The final parameters were: spray voltage of 4.0 kV, capillary temperature of 275 °C, heater temperature of 300 °C, sheath gas flow rate of 35 psi and auxiliary gas flow rate of 10 (arbitrary units). In addition, capillary voltage of 47.5 V, tube lens voltage of 186 V and skimmer voltage of 18 V were used. Nitrogen (purity > 99.999%) was employed as sheath gas, auxiliary gas and collision gas. The mass range was m/z 400-1,500 in full scan acquisition mode. The resolution was 50,000 (m/z 200, FWHM) at a scan rate 2 Hz, the automatic gain control (AGC) was set as "balanced" (1e 6 ) with a maximum injection time of 250 ms. The data was processed with Xcalibur 2.2 SP1 software (Thermo Fisher Scientific, Bremen, Germany).

Automatic identification/quantification were performed. The peaks were extracted from the chromatogram using the exact mass of both [M+NH 4 ] + and [M+Na] + diagnostic ions, the mass accuracy (±10 ppm extraction window), and the retention time window. In addition to HRMS and accuracy parameters for identification, in the present study, to be confident of the identification and the proposed elemental formulae, the following restrictive criteria were applied: elements considered were restricted in accordance with CTXs molecular formulae and adduct signals [C 55 to 70, H 64 to 110, O 11 to 25, N 0 to 1, and cations (Na) 0 to 1]; the isotopic pattern was matched and the charge, the ring double bond equivalents (RDBEs) and nitrogen rule were taken into account. Additionally, the monoisotopic pattern (M+1 ion) of these signals was used to assist in the further confirmation of the toxin's identity. Therefore, in total four diagnostic signals were used for toxin identification. The relative ion intensities between [M+NH 4 ] + , [M+Na] + and their M+1 ions were calculated and matched taking into account a tolerance according to the EU Decision 2002/657/EC. The toxins in study are characterised by a vast amount of carbon atoms, leading to relative abundances of the [START_REF] Hamilton | Multiple ciguatoxins present in Indian Ocean reef fish[END_REF] C isotopic ion higher than 65%. This high sensitivity of M+1 ion render these signals a high identification potential. The combination of high resolution, AMM and restrictive criteria was crucial for identification of both targeted and unknown compounds, as well as precise quantification of analytes. An external standard calibration was carried out from 12.5 to 100 ng/mL using P-CTX-1 and showed good linearity (R 2 = 0.996), with the LOD being 1.25 ng/mL. Due to the lack of proper analytical standards for all CTX congeners, in order to calculate concentrations of CTX analogues it was assumed that related analogues would give a similar response to that obtained with the P-CTX-1 standard.

Figure 1 .

 1 Figure 1. Toxicity of stomach fractions by cell-based assay (CBA). Toxicity was estimated according to the cell viability obtained after exposing cells to 0.27 µL of each fraction/mL. Error bars represent standard deviation (SD) values for 3 replicates (n = 3).

Figure 2 .

 2 Figure 2. Evidence for the presence of of ciguatoxins (CTXs) and gambieric acid D (GA D) in shark tissues. (a) Extracted ion chromatogram of I-CTX-1&2 [M+Na] + at 1163.6125 and HRMS exact mass spectra of (b) I-CTX-1&2 [M+Na] + at 1163.6125 and (c) I-CTX-5 (C 62 H 90 O 19 ) [M+Na] + at 1161.6070, in fraction F12 from stomach; (d) extracted ion chromatogram of I-CTX-3&4 ([M+Na] + at 1179.6084 and HRMS exact mass spectra of (e) I-CTX-3&4 [M+Na] + at 1179.6084 and (f) I-CTX-6 (C 62 H 90 O 20 ) [M+Na] + at 1177.5910, in stomach crude; (g) extracted ion chromatogram of GA D [M+NH 4 ] + at 1216.7354 and (h) HRMS exact mass spectra of GA D [M+NH 4 ] + at 1216.7354, in flesh crude extract, respectively.

LC-ESI-HRMS (µg P-CTX-1 equiv./kg tissue)

  C 62 H 92 O 19 NH 4 ] + and [C 62 H 92 O 19 Na] + ): 1158.6571, 1158.6606, <5.49 ppm and 1163.6125, 1163.6148, <4.93 ppm, respectively; and ii) I-CTX-3&4 ([C 62 H 92 O 20 NH 4 ] + and [C 62 H 92 O 20 Na] + ): 1174.6540,

	MBA (µg	CBA (µg
	P-CTX-1	P-CTX-1
	). All analogues were confirmed using their theoretical accurate mass (m/z), measured m/z, and mass accuracy extract equiv./kg tissue) equiv./kg tissue) I-CTX-1&2 I-CTX-3&4 Σ I-CTXs flesh n.q. 0.06 n.d. n.d. n.d. stomach 83 92.78 6.54 9.74 16.28 fin 1 -0.12 ---fin 2 -0.79 n.d. n.d. n.d. (ppm): i) I-CTX-1&2 ([Crude fin 3 -0.17 ---

Table 1 .

 1 Concentration of P-CTX-1 equiv./kg tissue in crude stomach, flesh and fin extracts as determined by mouse bio-assay (MBA), Neuro-2a cell-based assay (CBA) and liquid chromatography coupled to high resolution mass spectrometry (LC-ESI-HRMS).

		CBA (%	LC-ESI-HRMS (% P-CTX-1 equiv.)
		P-CTX-1			Σ
	Fractions	equiv.)	I-CTX-1&2	I-CTX-3&4	I-CTXs
	F8	0.23	n.d.	n.d.	n.d.
	F9	5.15	n.d.	5.67	5.67
	F10	5.80	n.d.	29.66	29.66
	F11	9.37	13.87	15.75	29.62
	F12	8.05	16.12	3.86	19.98
	F13	1.79	n.d.	n.d.	n.d.
	F14	1.44	n.d.	n.d.	n.d.
	F15	2.30	n.d.	n.d.	n.d.
	F16	0.62	n.d.	n.d.	n.d.
	F17	0.29	n.d.	n.d.	n.d.
	F18	0.27	n.d.	n.d.	n.d.
	F19	0.33	n.d.	n.d.	n.d.
	F20	0.23	n.d.	n.d.	n.d.
	F21	0.19	n.d.	n.d.	n.d.
	F22	0.12	n.d.	n.d.	n.d.

Table 2 .

 2 Distribution

of ciguatoxins (CTXs) after fractionation of the stomach crude extract. Percentages of P-CTX-1 equiv. recovered in each fraction in relation to the P-CTX-1 equiv. injected, estimated by the Neuro-2a cell-based assay (CBA) and liquid chromatography coupled to high resolution mass spectrometry (LC-ESI-HRMS). 1174.6565, <6.31 ppm and 1179.6084, 1179.6146, <5.89 ppm, respectively.

  5 ([C 62 H 90 O 19 NH 4 ] + and [C 62 H 90 O 19 Na] + ): 1156.6414, 1156.6479, 5.57 ppm and 1161.5968, 1161.6030, 5.29 ppm, respectively; and ii) I-CTX-6 ([C 62 H 90 O 20 NH 4 ] + and [C 62 H 90 O 20 Na] + ): 1172.6496, 1172.6381, -1.44 ppm and 1177.5918, 1177.5953, 3.03 ppm in fraction F12 from stomach, respectively. Full HRMS exact mass spectra of both new I-CTXs in Fig.

Scientific RepoRts | 7: 8240 | DOI:10.1038/s41598-017-08682-8

Acknowledgements

We kindly acknowledge Dr. Bernard Reche, the veterinarian in charge of the shark autopsies in La Réunion and who confirmed the viscera sample was stomach, and also Erwan Lagadec (CRVOI) for performing the DNA extraction. We also thank the World Health Organization (WHO) for facilitating the transfer of samples from Madagascar to Reunion Island. The authors acknowledge Dr. Chris Rodgers for his critical review of the manuscript and Dr. Melissa Dunkle for assistance with the English. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under the ECsafeSEAFOOD project (grant agreement no. 311820) and from CERCA/Generalitat de Catalunya program.

Author Contributions

Additional Information

Supplementary information accompanies this paper at doi:10.1038/s41598-017-08682-8

Competing Interests: The authors declare that they have no competing interests.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Louison Suard

Eric Collias

Anatole Danto

Eco-anthropology in Lower Loire: waterfowl hunting and related knowledges

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.

II. How do hunters mobilize their knowledges ?

Knowledges are necessary to hunt on Loire's Islands. Tides, flood, low water, wind and atmospheric pressure modify how water comes over the different islands.

Hunters could easily get stucked and would have to wait the ebb tide.

All those things needed to go hunting are known the day before or during the day. Hunters do exchange informations between them about weather and so the game via speaking but also via social networks. They use too weather websites made for other activities like surfing or fishermen. Indeed, weather has a direct impact on game.

Waterfowl hunters would also not use call-birds the same way to attract wild individuals according to the month. At the opening birds are what we could call « naive ». Individuals do not mistrust groups of individuals calling on a pond and come easily. At that period, hunters are not afraid to use many call-birds (more than 5). Later in the season, individuals mistrust those ponds too noisy and avoid those areas. Like many hunters said « le canard n'est pas con » (« duck isn't stupid »). Hunters use then more fake call-birds in plastic, knowing waterfowl are gregarious during migration. Some hunters also consider the influx of new individuals thanks to migration (there are websites to follow those migrations) and so use real call-birds again. Each hunter has its own strategy to know when use them, in which proportion (fake or real call-birds) and according to the environment where they hunt.

Eco-anthropology in Lower Loire: waterfowl hunting and related knowledges

I. Introduction

Waterfowl hunting is the hunt of some bird species who need wetlands to live, for their reproduction or for food. Three groups are concerned : Anatidae, Rallidae and wadind bird. In France, it is 37 waterfowl species that can be hunted from the 21 st august to the 31 st january (except some species that are hunted longer).

Waterfowl hunting takes place in wetlands, like Loire estuary (France, Pays de la Loire, Loire-Atlantique ; map 1) and the Lower Loire where wetlands are common (Grande Brière Mottière, Guérande swamps and other ones). We took interest for one zone delineated in map 1. That zone is formed of different islands, created by sediments accumulation, result of the tides. Those islands are a great place to hunt waterfowl. Ponds, created by hunters or farmers are disseminated on those islands and contribute to the attractiveness of this environment.

We consider waterfowl hunters in their environment as Tim Ingold defines it (T. Ingold, 2012) : i) « environment » depends on the person considering it, ii) it is not something finished, it is something continually changing, made up by all living beings sharing that environment, iii) « environment » can not be confounded with « nature », to consider « nature » is putting it aside from living beings.

We have different aims. We want to learn which knowledges are necessary to hunt waterfowl, how hunters mobilize them and how do they covey them. We also want to know to compare thoses knowledges to naturalists's, according to the dualism exposed in human sciences opposing « traditional » and « modern » knowledges.

To learn about how waterfowl hunting happens in this area, we adopted an qualitative method with semi-directive interviews, field survey and participant observations. Hunting stories about Lower Loire have also been analysed, like Jean de Witt stories. We also decided to explore three differents themes about waterfowl hunting we will expose here.

III. How those knowledges are transmitted ?

We thought that maybe hunters, as a traditional activity, would transmit their knowledges between members of a same family, from father to son, or uncle to nephew. That hypothsis is not true. Among all the hunters we met, however their age, hunters have learnt hunting in different ways. Some of them started alone, having « fun » when they were younger on those islands and started hunting illegally, because they did not have legal age to hunt. Some of them learnt with a member of their family when they were younger, coming over a day to see how it works. Rarely, some hunters have discovered hunting with friends.

But however they discovered waterfowl hunting, knowledges are also transmitted among hunters on the field when they meet. Some of them consider it is important to train other hunters and to share their knowledges so waterfowl hunting could perpertuate.

IV. Hunters and naturalists knowledges

Both hunters and naturalists have knowledges about the field, the species and their behaviours but those knowledges are differents because hunters and naturalists do not have the same interests.

Consequences of migrations, the weather (rain, wind) are known by both. Hunters knows things allowing them to hunt. For example, they know which traces leaves a Common Teal (Anas crecca, Linnaeus, 1758) when it eats somewhere. Common Teals eat at night, so hunters know they have to come there to hunt Common Teals in the evening when they find thoses traces. This behaviour is not something known by the naturalists, excepts for the ones who are also hunters. Both naturalists and hunters have their own knowledges necessary to their approch of the animal, even though there is common features. Those common features can be observed through different phases : to attract birds -to catch them -wonder observing birds -to know species behaviours and having favourite species.

V. Conclusion

Hunters have their own knowledges to hunt waterfowl ; those knowledges are intimately linked to the environment they hunt in. Hunters imagine how game is going act and match their strategy according to it.

Those knowledges, hunters are not aware of them. What they do, they do it « au pif » (that could be translated as « guessworking » or « randomly »), those are empirical knowledges. They get them through a practice of their environment. Traditional knowledges are usually compared to « modern knowledges »: without considering this dualism veracity, it does explain in part all the conflicts between those two groups. With the modification of the human-animal (C.Pelluchon, 2017) relationship, we can interrogate ourselves about hunting future. This modification also interrogate hunters themselves about the future of the activity, being more and more threatened by animal welfare associations. Getting to know better hunting in France is also a chance to learn about the relationship Human-Animal and so human-environment as it is in occident. It is also an opportunity to better understand what is/could be naturalism as Descola (P. Descola, 2005) described it.

Image 1 :

 1 Waterfowl hunting in the evening in front of a pond, Ile Pipy. ©Louison Suard Image 2: Ile de Pierre-Rouge during the day with a pond, used for cattle and for waterfowl. ©Louison Suard SFECOLOGIE 2018 International Conference on Ecological Sciences October 22 th -25 th Rennes

  Louison Suard 1 , Eric Collias 2 , Anatole Danto 3 1, Master Anthropologie sociale et ethnologie, EHESS ; 2, Ecographe, Saint-Nazaire ; 3, CNRS, UMR ARENES, UMR CEBC, RTPi ApoliMer Corresponding author: Louison Suard, louison.suard@gmail.com


Fishing shrimp in the Wadden Sea: the extinction of a traditional practice in the Frisian area of Bremerhaven (Weser mouth) Anatole Danto, Henning Siats

Threatening social and environmental changes

Social changes:

▪ For several years, the inhabitants of the Weser estuary have gradually abandoned the primary sector in favour of work in the port of Bremerhaven. ▪ The fishing sector has experienced a significant decline. Sledding, which is physically exhausting, was one of the first to almost disappear. The rotation of traps twice a day (at each tide) is indeed a strong constraint. Shrimp shelling in Germany has also become too expensive in terms of labour. Only two fishermen still practice this fishing technique on a daily basis, in Wremen and in the Ems estuary.

Environmental changes:

▪ At the same time, the coastal environment of the Wadden is constantly changing, and is facing both the increase in the surface of the port area, to the detriment of the mudflat, and the emergence of new uses at sea (RME, drilling, etc.). These changes affect both the shrimp population and fishing areas, which sometimes become impassable.

The « museum setting » Birth and objectives of the Wremen's Museum für Wattenfischerei:

▪ The museum was established in Wremen in 1991 ;

The Wremer Heimatkreis had set itself the goal of establishing and operating a museum in which the Wadden Sea fishery in its different species and methods and the living conditions of the fishermen and their families in the centuries are to be documented. ▪ The museum still works today with the fishermen of Wremen, both by boat and sled, and contributes to the conservation of the particular fishing technique once practised in Wremen. ▪ The museum has important ethnographic, iconographic and filmographic collections, highlighted within a museum space, and inventoried. ▪ The museum also helps researchers in their scientific work, in particular in its archives, or during field surveys.

Oceans Past VII Conf. / AWI / German Maritime Museum / Bremerhaven, GERMANY / 22 th -26 th of October 2018

Abstract

For millennia, coastal societies in the Wadden Sea have practiced environmental food uses along the coasts of this semi-enclosed sea. The regional Germanic peoples (Frisians, Lower Germans, Dutch, Danish Jutlanders, etc.) have established important hunting, fishing, agricultural and forestry techniques in these territories (Danto, 2017). Some species, emblematic of the European fishing bestiary, are particularly sought after, such as the North Sea shrimp (Crangon crangon). Various predation techniques have been put in place to capture it. While some are widely practiced (such as beam trawling), others are more or less threatened due to a lack of sufficient practice, with loss of local knowledges (Ingold, 2012). Shrimp fishing on horseback, particularly in Belgium, has been classified as a UNESCO World Heritage, giving it heritage visibility. In contrast, sled fishing has only two or three peoples, concentrated in Germany, who are the last to perpetuate a centuries-old tradition of responsible and sustainable fishing for the Wadden Sea.

Key words

Eco-anthropology, Ethnoichtyology, Fisheries, Coastal communities, LTK, Nature/Culture, Shrimp Crangon crangon, Weser mouth, Wremen, Wadden Sea, North Sea, Heritage.

The shrimp

Shrimp fishing in the North Sea is particularly targeted at the grey shrimp, Crangon crangon. The population of grey shrimp in the North Sea is very large, thanks to a particular ecosystem, that of the great mudflat of the North Sea. It is one of the largest in the world, fed by many continental rivers (Rhine, Elbe, Scheldt, Meuse, ...), and has a particular tidal regime on the tide area (Watt).

Fishing shrimp in the Wadden Sea: the extinction of a traditional practice in the Frisian area of Bremerhaven (Weser mouth)

Anatole DANTO 1 , Henning SIATS 2 The « funnel », added value in Watt ▪ The shrimp traps in the Wadden Sea are inserted into large wicker funnels, forming the fishing gear (Spann). The two assembled elements are placed horizontally on the muddy foreshore, using chestnut stakes.

Traditionnal Reuse of Wremen area © Anatole Danto, 2017.

The sled

▪ The sled provides access to the fishing areas where the gear is placed. They are generally built for the year, in oak, directly by the fisherman. They are pulled by dogs, with slightly webbed legs (Labrador for example), rather crossed ("bastards", more adapted to the Watt). Sleds allow fishermen to travel several kilometers at each tide to reach their fishing territory, which consists of a tidal channel, in the heart of the mudflat.

The sled of Erhard Djuren, in Wremen © Anatole Danto, 2015. Crangon crangon in Wremen, after fishing, © Anatole Danto, 2017.

Eco-anthropology

The shrimp fishing

In the Wadden Sea, different fishing and hunting techniques have been in place for many years, targeting fish, shellfish, crustaceans, marine mammals or seabird (Meier, 2003). Shrimp were initially caught using fishing gear such as traps (Reuse) or small nets in the riparian fishery.

Over time, there has been a specialization of gear in shrimp predation (adaptation of meshes, positioning of gear), followed by major technological developments, making it possible to fish shrimp in much greater quantities, first by moving further forward on the foreshore during the low seas (this will be the invention of the sled on the Watt), then thanks to naval advances (first offshore vessels, discovery of the trawl, then the beam trawl, motorization of vessels).

Some of these advances are based on empirical analysis of fishermen or fish merchants, others are based on scientific research. Sometimes they seek to go beyond an overly rigid regulatory framework (Bavinck & Hoefnagel, 2005).

  1 : CNRS PhD candidate / International research network ApoliMer : political anthropology of the Sea / Corresponding author : anatole.danto@orange.fr UMR 6051 ARENES : Research center on policy action in Europe, Institute of political studies of Rennes / UMR 7372 CEBC : Chizé Centre for Biological Studies 2 : Director of the Museum für Wattenfischerei of Nordseebad Wremen Shrimp sled and trap fishing Traps as fundamental technology ▪ Traps are among the oldest known fishing gears. They are found in many archaeological excavations all over the world.Initially, they were built entirely of vegetable materials. In the Wadden Sea, the traditional trap is made of wicker, with fishermen owning their own willow orchard near their farm.Traditionnal trap of Wremen village © Anatole Danto, 2017.

  ., Hoefnagel H., 2005, "Capituler, esquiver, protester -Face aux interventions de l'Etat dans le secteur de la pêche, les pêcheurs ont adopté diverses stratégies en mer des Wadden", in SAMUDRA, n°41, pp. 17-23. -Danto A., 2017, Sociétés littorales et usages traditionnels du Watt depuis le Moyen-Âge, MA thesis, University of South Brittany, 202 p. -Ingold T., 2012, "Culture, nature et environnement", in Tracés, Revue de sciences humaines, vol. 22, pp. 169-187. -Meier D., 2003, "Landschaftsentwicklung und historische Nutzung der Nordseeküste", in Anthropogene Einflüsse, pp. 161-166.

Ehrard

  Djuren during fishing in Wremen coast © Anatole Danto, 2017.. Fishing trip in Wremen coast © Anatole Danto, 2017..


Regarding the impacts on the vegetal cover, as the PUU invest largely in access infrastructure it is more likely that livestock load is more evenly distributed. However, as there is no monitoring outputs are questionable.

The PUUs are one of the few user based institutions in a set up where topdown approach dominates which hinders collaboration.

The PUUs because of their weak legitimacy rely on pasture users and need to make sure that no stakeholder group is harmed by their measures. In this sense, they strengthen resilience by increasing intercommunity interactions. Community-Based Pasture Management allows the inhabitants to be actively part of the formal management, with their perception and knowledge of the environment.

Pasture Users Unions : giving the users responsibility?

All pasture users in one rural municipality are members of the PUU. However, governance is made difficult because of the of the institutions and stakeholders playing different roles : the users, the PUU, the executive body and the rural municipality.

Grass-root decision-making in heterogenous communities

The end of pasture lease Shepherds are more likely than livestock owners to be excluded from the decision making as they live outside of the villages, are mobile and were not involved in the implementation. However, they are interested in the actions of the PUU and try to influence the management plan.

The previous pasture management model based on lease was inefficient in raising funds. Nowadays, pasture user pay a fee per livestock head, defined localy. PUUs also receive funds from international organizations.

Pasture Degradation and livestock mobility

The community-based pasture management was partly adopted to fight pasture degradation which occurred because of the reduction of livestock migrations. It was expected that it could be overcome by involving the users, and through financial support of international organizations, PUUs would have the financial capacity to rehabilitate Soviet infrastructure and to develop management plans.

Degradation as the reflect of conflicting concepts

Infrastructure : the core of pasture management?

PUU do not have the capacities nor are legitimate to allocate pastures. PUU focus on the rehabilitation of Soviet infrastructure of access as it encourages longer transhumances, is easy to implement for PUU, and allows to rally support from pasture users.

Shepherds setting up their yurt for the first time on spring pastures. The pasture they use was allocated by other shepherds nearby and not by the PUU

The lack of unified concept of degradation and of methodology to measure it, hinders the capacity of PUU to monitor pasture quality. Pasture management plans according to pasture conditions to regulate the use are not developed.

Coordinating in a scattered management

PUUs administer only 70% of the pastures of the country, the rest being under the management of Forestry Mangement Units. However, managers need to deal with a seasonal use of pasture which does not always fit to that administrative borders..

Innovative mechanisms for inter-PUUs cooperation

PUUs coordinate their actions regarding the price setting for pasture users from other PUUS, the dates of migration and the number of animals allowed. These issues have potential for conflicts, however, the creation of PUUs associations at district level at their initiative shows positive results.

Forestry Management Units and Pasture User Unions: overcoming conflicting understanding of the environnement

The Forestry Management Units function differs from PUUs as their official objective is nature conservation. However, because of lack of fundings, pasture lease is one of their major source of income.

  Community-Based Pasture Management in Kyrgyzstan What impacts on the resilience of local communities? Pasture User Unions (PUU) were introduced in Kyrgyzstan in 2009. Although Water User Associations already existed, it was the first time community-based management was initiatied on pastures for the whole centralasian region. Pastures are at the cross-point of major economical, ecological and social issues for Kyrgyzstan. Based on the theory of the Commons of Ostrom (1990) and work on territorial resilience of Aschan-Leygonie (2001), we investigate how the communitybased pasture management affects the resilience of local communities. Irène Mestre, Université Jean Moulin-Lyon 3, Centre de Recherche en Géographie et Aménagement, UMR 5600 Environnement-Ville-Société, with the support of the Mountain Society Research Institute, the French Institute for Central Asian Studies and the Centre for Development and Environment

  The price setting for pasture users from other rural municipalities The sharing of pasture use fee amongst PUUsThe dates of transhumance The control mechanisms

		Pasture Committee -executive body	• Works out the management plan and the use plan,
		Local deputies Members of the Pasture	• Controls the implementation of management measures
		Users Association
		Pasture Users Union All pasture users, for all types of use	• Elects • Adopts management plans with the municipality council
			• Pays for pasture use
		Sedentary livestock owners	Mobile shepherds
	Rural Municipalities on: The management plan and the budget allocation The share of budget transferred to the rural municipality	PUU needs to coordinate with	Forestry management units on: Prices for pasture users and sharing of pasture fees between FMU and PUUs, Dates, The number of animals allowed, Responsibility for infrastructure leading to FMU and PUU pastures
		Other PUUs on: