Pierre-Yves Pascal
email: pypascal@univ-ag.fr

Hidetaka Nomaki

Yosuke Miyairi

Yusuke Yokoyama

The use of radiocarbon to evaluate the trophic role of geothermal bacteria in shallow hydrothermal water ecosystem

Keywords: δ 13 C, δ 15 N, Δ 14 C, diet, sulfur bacteria, food web

Stable isotopes such as δ 13 C and δ 15 N are routinely used in trophic ecology as isotope values are derived from diet and recorded subsequent fractionation in consumer tissue.

However, this approach necessitates to estimate a priori the fractionation between food source and consumer leading to potential uncertainties. In this context, the development of additional biomarkers enables to have better resolution on feeding habits. Dissolved Inorganic Carbon (DIC) from volcanic activity and bacteria using this DIC presents a strongly depleted natural radiocarbon abundances (Δ 14 C). Through its activity, the geothermal plant of Bouillante in Guadeloupe (French West Indies) releases sulfur bacteria in shallow environment of the Bay. A previous study reveals ingestion of those bacteria by opportunist species (sea urchin and fish species). In the present study, ten species with different diet and feeding mode were sampled close to the release channel of the geothermal plant and in a control station in order to simultaneously determine their stable isotopes (δ 13 C and δ 15 N) and radiocarbon compositions. Compared to models using Δ 14 C data, models using δ 13 C and δ 15 N data underestimate the role of bacteria in diet of urchin and fish species whereas this role is overestimated for all other species. Compared to previous models, radiocarbon would give more realistic and reliable results than the classical use of stable isotope. This study confirms the utility of radiocarbon in food web ecology, particularly at the ecosystems having food sources with contrasting Δ 14 C values.

1) Introduction

Since more than three decades, carbon and nitrogen isotope measurements (δ 13 C and δ 15 N values) are routinely used to determine trophic interactions in marine and terrestrial environments [START_REF] Peterson | Stable isotopes in ecosystem studies[END_REF]. They constitute the most used tool in trophic ecology as isotope values are derived from diet, and are recorded in consumers tissues [START_REF] Fry | Stable isotope ecology[END_REF].

Fractionation is the selective retention of heavy isotopes during metabolic reactions within the body of consumers. Difference in δ 13 C signature between food sources and consumers is usually small (<1‰), allowing accurate determination of diet composition [START_REF] Mccutchan | Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur[END_REF]) whereas δ 15 N in consumer are usually enriched by about 3‰ relative to their diet allowing identification of food resources and trophic level [START_REF] Minagawa | Stepwise enrichment of 15 N along food chain: further evidence and the δ 15 N and animal age[END_REF][START_REF] Post | Using stable isotopes to estimate trophic position: models, methods, and assumptions[END_REF]. The fractionation needs to be estimated a priori in order to quantify food web relationships. However, this fractionation can vary in consumers according to their phylogenic group (e.g. vertebrates, invertebrates etc.), their diet (e.g. herbivores, carnivores etc.), their physiological states and environmental conditions [START_REF] Vander Zanden | Variations in δ 15 N and δ 13 C trophic fractionation: implications for aquatic food web studies[END_REF][START_REF] Caut | Variation in discrimination factors (Δ 15 N and Δ 13 C): The effect of diet isotopic values and applications for diet reconstruction[END_REF], Cabanillas-Téran et al. 2019). All those uncertainties can result in error of source contribution estimated by model hindering the use of stable isotope in foodweb studies [START_REF] Vander Zanden | Variations in δ 15 N and δ 13 C trophic fractionation: implications for aquatic food web studies[END_REF]. Isotopic pools with differing origins, such as body reserves, muscle protein, and direct food intake can have non-equal contributions to tissue formation complicating determination of trophic role of sources [START_REF] Fry | Stable isotope studies of pink shrimp (Farfantepenaeus duorarum Burkenroad) migrations of the southwestern Florida shelf[END_REF][START_REF] Ayliffe | Turnover of carbon isotopes in tail hair and breath CO 2 of horses fed an isotopically varied diet[END_REF]. Stable isotope can also present limitations when isotope ratios of potential sources are overlapping and/or when they present high variabilities over short spatial or temporal scales. In order to increase discrimination between potential food sources, stable isotope can be used as tracers during experiments with prey artificially enriched in heavy isotopes. This approach is adapted to evaluate ingestion of small size food items such as bacteria [START_REF] Van Oevelen | The fate of bacterial carbon in sediments: modeling an in situ isotope tracer experiment[END_REF][START_REF] Pascal | Trophic role of large benthic sulfur bacteria in mangrove sediment[END_REF], microphytobenthos [START_REF] Moodley | Bacteria and foraminifera: key players in a short-term deep sea benthic response to phytodetritus[END_REF][START_REF] Como | Stable isotope as tracers can reveal resource allocation in juvenile golden gray mullets (Liza aurata, Risso, 1810)[END_REF]) and meiofauna [START_REF] Pascal | Meiofauna versus macrofauna as food ressource in a tropical intertidal mudflat[END_REF]. Enrichment experiments can be conducted in situ even in remote environment such as deep sea [START_REF] Moodley | Bacteria and foraminifera: key players in a short-term deep sea benthic response to phytodetritus[END_REF][START_REF] Nomaki | Different ingestion pattern of 13 C-labeled bacteria and algae by deepsea benthic foraminifera[END_REF]). However, they are limited to small size preys and can present bias due to potential modification of feeding behavior in artificial conditions of experiments. Other approach to face shortcoming of stable isotopes is to use fatty acid biomarkers in addition to stable isotopes [START_REF] Jaschinski | Carbon sources and trophic structure in an eelgrass Zostrea marina bed, based on stable isotope and fatty acid analyses[END_REF][START_REF] Leduc | A multi-method approach for identifying meiofaunal trophic connections[END_REF][START_REF] Lebreton | Trophic importance of diatoms in an intertidal Zostera noltii seagrass bed: evidence from stable isotope and fatty acid analyses[END_REF]. The use of fatty acid can be challenging as it requires several criteria such as the unique and high abundance of fatty acids in potential food sources and the absence of neo synthesis of fatty acid in consumers [START_REF] Chamberlain | Fatty acid composition and change in Collembola fed differing diets: identification of trophic biomarkers[END_REF]. All methods conventionally used in trophic ecology and mentioned above presents potential shortcomings and limitations according to environments and organisms studied. The best way to increase reliability of results from food web studies is consequently to use simultaneously several methods. In this context, the development of additional biomarkers will help to answer different research question in trophic ecology.

Radiocarbon ( 14 C) is the radioactive isotope of carbon continually produced in the earth's upper atmosphere by cosmogonic radiation. Due to the radioactive decay with the half-life of 5730 years, the concentration of 14 C (Δ 14 C) vary among the earth's different carbon reservoir and often presents a wider dynamic range than δ 13 C in certain ecological contexts [START_REF] Larsen | Radiocarbon in ecology: Insights and perspectives from aquatic and terrestrial studies[END_REF]. Unlike δ 13 C and δ 15 N values, the Δ 14 C constitute a direct reflection of a specific source and is not affected by variable fractionations according to metabolic pathways.

Due to this absence of discrimination, radiocarbon can constitute robust tracer of food source with an advantage over stable isotopes [START_REF] Ishikawa | Natural C-14 provides new data for stream food-web studies: a comparison with C-13 in multiple stream habitats[END_REF][START_REF] Larsen | Radiocarbon in ecology: Insights and perspectives from aquatic and terrestrial studies[END_REF][START_REF] Nishida | Novel reverse radioisotope labelling experiment reveals carbon assimilation of marine calcifiers under ocean acidification conditions[END_REF]. Historically, measurements of 14 C require substantial sample mass [START_REF] Williams | Dietary carbon sources of mussels and tubeworms from Galapagos hydrothermal vents determined from tissue 14 C activity[END_REF]) but recent technical improvements of analysis (e. g. [START_REF] Yokoyama | A single stage Accelerator Mass Spectrometry at the Atmosphere and Ocean Research Institute, The University of Tokyo[END_REF][START_REF] Yokoyama | Efficient radiocarbon measurements on marine and terrestrial samples with single stage Accelerator Mass Spectrometry at the Atmosphere and Ocean Research Institute[END_REF]) allow measurements in small size organisms like meiofauna [START_REF] Nomaki | Nutritional sources of meio-and macrofauna at hydrothermal vents and adjacent areas: natural-abundance radiocarbon and stable isotope analyses[END_REF] and in selective compounds such as microbial lipid [START_REF] Pearson | Bacterial incorporation of relict carbon in the hydrothermal environment of Guaymas Basin[END_REF]) and amino acid [START_REF] Blattmann | Liquid chromatographic isolation of individual amino acids extracted from sediments for radiocarbon analysis[END_REF]). Radiocarbon analyses are more time consuming and complicated than stable isotopes ones, though there are several advantages including i) it requires less replications as 14 C is less variable [START_REF] Ishikawa | Natural C-14 provides new data for stream food-web studies: a comparison with C-13 in multiple stream habitats[END_REF], ii) it requires less precision than in 14 C archeological studies and iii) it becomes gradually more affordable and for those different reasons, radiocarbon will likely be more used in trophic ecology in coming decade [START_REF] Larsen | Radiocarbon in ecology: Insights and perspectives from aquatic and terrestrial studies[END_REF].

In marine environment, variation of radiocarbon according to geographic location allow the study of the role of currents in food availability [START_REF] Ishikawa | Influences of ocean currents on the diets of demersal fish communities in the Western North Pacific Revealed by their muscle carbon and nitrogen isotopic compositions[END_REF]) and feeding behavior of migrating marine life from fish [START_REF] Yokoyama | Radiocarbon as a marine ecological tool box[END_REF] to whale [START_REF] Eisenmann | Radiocarbon as a novel tracer of extra-antarctic feeding in southern hemisphere humpback whales[END_REF]). 14 C slowly decays over time with a half-life of 5730 years and allow evaluation of trophic role of i) recent food items from surface ocean and for bottom dwelling fauna [START_REF] Rau | Upwelling and the condition and diet of juvenile rockfish: a study using 14 C, 13 C and 15 N natural abundances[END_REF][START_REF] Purinton | 14 C as a tracer of labile organic matter in Antarctic benthic food webs[END_REF][START_REF] Guillemette | Old before your time: ancient carbon incorporation in contemporary aquatic foodwebs[END_REF] and ii) trophic role of old food items from oil reservoir [START_REF] Chanton | Examination of coupling between primary and secondary production in a river-dominated estuary: Apalachicola Bay, Florida, USA[END_REF][START_REF] Wilson | Tracing the intrusion of fossil carbon into coastal Louisiana macrofauna using 14 C and 13 C abundances[END_REF], upwelling [START_REF] Berkman | Pre-bomb radiocarbon and the reservoir correction for calcareous marine species in the Southern Ocean[END_REF] and deep-sea hydrothermal vent activities [START_REF] Williams | Dietary carbon sources of mussels and tubeworms from Galapagos hydrothermal vents determined from tissue 14 C activity[END_REF].

Deep sea environments are isolated from the contemporary atmosphere and are depleted in 14 C relative to surface ocean. In deep sea hydrothermal vent, chemoautotrophic microbes typically exhibit depleted 14 C concentrations as their inorganic carbon source comes from 14 C-depleted bottom-water [START_REF] Stuiver | Abyssal water carbon-14 distribution and the age of the world oceans[END_REF]) and/or 14 C depleted CO2 or CH4 released through volcanic activity [START_REF] Bruns | Regional sources of volcanic carbon dioxide and their influence on 14 C content of present-day plant material[END_REF][START_REF] Williams | Dietary carbon sources of mussels and tubeworms from Galapagos hydrothermal vents determined from tissue 14 C activity[END_REF]). In such chemosynthesis-based ecosystems, radiocarbon can be used more efficiently to evaluate trophic role of chemolithoautotroph microbes in deep sea [START_REF] Nomaki | Nutritional sources of meio-and macrofauna at hydrothermal vents and adjacent areas: natural-abundance radiocarbon and stable isotope analyses[END_REF].

The aim of the present study is to evaluate the potential of radiocarbon as tracer of chemoautotroph bacteria in coastal tropical environment. In Guadeloupe Island (French West Indies), a plant pumps geothermal water rich in H2S from deep reservoir in order to produce electricity. This water is then cooled with sea water before being released into the sea.

Environmental conditions in the discharge channel support development of sulfur-oxidizing bacteria. A previous study using i) fish counts according to plant activity and ii) stable isotope compositions (C, N, S) of potential consumers of bacteria, reveals an ingestion of sulfur bacteria by sea urchin and fish species whereas this trophic role was not detected for suspension feeders and omnivorous predators [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF]. Sulfur-oxidizing bacteria presents high concentration of Hg and bacterial consumers (urchin and fish) bioaccumulate more Hg in Bouillante Bay than in adjacent environment confirming their active ingestion of bacteria (Ruiz et al. In prep). In order to evaluate the potential of radiocarbon as a tracer in food web, we tested 10 species previously analyzed with various feeding mode and different contribution of sulfur bacteria in their diet, where sampled close to the release channel of the geothermal plant and in a control station. The simultaneous determination of stable isotopes ( 13 C and 15 N) and radiocarbon ( 14 C) compositions of organisms allowed to compare results of modeled contribution of bacteria in their diet and the utility of radiocarbon tracer.

2) Material and method

2.1) Study area

The study area is located on the west coast of Basse Terre Island in Guadeloupe in French West Indies, which belongs to the active Lesser Antilles volcanic arc (Fig. 1). The multiple hot springs, steaming grounds and fumaroles characterizing this region explain the name of the town "Bouillante", which means "boiling" in French. Infiltration of rainwater (40%) and sea-water (60%) fills several fractured zones between 150 and 1100 m forming a reservoir of 30 million m 3 with a temperature of 250-260°C [START_REF] Lachassagne | Hydrogeological model of a high-energy geothermal field (Bouillante area, Guadeloupe, French West Indies)[END_REF]). To exploit this source of energy, a geothermal plant without reinjection started producing electricity in 1986 exploiting water coming from a 340 m deep well [START_REF] Jaud | The Bouillante geothermal power-plant, Guadeloupe[END_REF].

Additional deeper wells (1000-1200 m) are actually used to produce 6-7% of Guadeloupe electricity consumption [START_REF] Demarcq | Situation and perspectives of the Bouillante geothermal power plant in Guadeloupe, French West Indies, Deep geothermal days[END_REF]. All residual water is mixed with pumped seawater in order to reduce the temperature to 45°C before being dumped at sea through discharge channel (Fig. 1). Geothermal fluid is characterized by a high concentration of dissolved H2S reaching 35-45 mg l -1 in the steam condensate [START_REF] Mas | Clay minerals related to the hydrothermal activity of the Bouillante geothermal field (Guadeloupe)[END_REF]. Chemical and temperature conditions are favorable for bacteria forming a thick white mat (6-7 mm) visible with naked eye and covering pebbles at the bottom of discharge channel. This bacterial mat is constituted by cyanobacteria (Pectonema sp) covered by white sulfur-oxidising bacteria of the genus Thiomicrospora [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF]. Due to strong current outflow (2.5 m 3 s -1 ), bacteria are regularly ripped off from pebbles, carried by current and used as food resource by urchin and fishes at the discharge channel outlet [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF]. Anse à la barque (AALB) was used as control station located 4.5 kms at the South of Bouillante Bay (Fig. 1) where no effects of such hydrothermal fluid and bacterial mat was recorded.

2.2) Sampling

Samples were collected in June 2020 while geothermal plant was active since a long period. For water sampling, 1) geothermal fluid was collected in Bouillante geothermal plant before its dilution with sea water and 2) surficial sea water was collected in AALB, 50 m away from the shore. Water samples were kept sealed and poisoned with HgCl2 before their analyze. Pebbles were collected from Bouillante discharge channel in order to scrap and sample their bacterial biofilm for Δ 14 C analyses. Stable isotopic compositions (δ 13 C and δ 15 N) of bacterial biofilm, sampled with similar protocol, over a long time period (2014)(2015) and previously analyzed were averaged for present study. All other samples were collected through free diving in Bouillante close to the water discharge outlet (with a distance from the outlet less than 5 m) and in AALB. Echinoderm (Diadema antillarum), polychaeta (Hermodice carunculata), bivalves (Spondylus tenuis), crustacean (Balanus sp.), cnidarian Pseudopterogorgia sp. and sponges (Aplysina fistularis and Iotrochota birotulata) were collected by hand. Fishes (Acanthurus bahianus and Abudefduf saxatilis) were speared. Four specimens of each species were taken from each site. A dissecting microscope was used to isolate muscles of the echinoderm, polychaeta, bivalves and fishes. For cnidarian, crustacean and sponges coarse tissue were sampled. Samples were frozen, freeze-dried and then acidified with 0.1M HCl to remove calcium carbonate and then weighed into precleaned silver capsules before analyse.

2.3) Stable isotopes (δ 13 C and δ 15 N)

Carbon and nitrogen isotopic composition were determined using an isotope ratio mass spectrometer coupled to an elemental analyzer (Flash EEA 1112-DELTA V Advantage ConFloIV System, Thermo Fisher Scientific). Isotope ratios were conventionally expressed as δ values in ‰ [START_REF] Coplen | Guidelines and recommended terms for expression of stable isotope ratio and gasratio measurement results[END_REF] relative to C and N international standards (respectively Vienna PeeDee Belemnite and atmospheric N2). For the machine calibration of the IRMS, NBS19 and Sucrose ANU were used for δ 13 C and IAEA N1 and IAEA N2 were used for δ 15 N measurements. Every 10 to 15 samples, isotopic compositions of working standards for δ 13 C C (Alanine: -19.6‰; Histidine: -10.7‰; Glycine: -33.8‰) and δ 15 N (Alanine: 1.58, 9.97‰, and 20.6‰) were measured.

2.4) Radiocarbon (Δ 14 C)

Corrected water samples were treated to extract Dissolved Inorganic Carbon (DIC) using the bubbling method [START_REF] Mcnichol | The rapid preparation of seawater CO 2 for radiocarbon analysis at the National Ocean Sciences Ams Facility[END_REF]. Namely, 4 ml of phosphoric acid were added to 250 ml of water, and bubbling was conducted for 15 min in a glass vessel filled with 60 mPa of helium gas to strip CO 2 liberated from DIC. Water vapor is released along with the CO 2 gas during this process, so 2 cryogenic traps containing ethanol cooled to -120 °C were used to remove the water vapor. The CO 2 was captured in a liquid nitrogen trap (Servettaz et al. 2019). The CO 2 collected was processed under the protocol described in [START_REF] Yokoyama | Relation between acid dissolution time in the vacuum test tube and time required for graphitization for AMS target preparation[END_REF][START_REF] Yokoyama | Efficient radiocarbon measurements on marine and terrestrial samples with single stage Accelerator Mass Spectrometry at the Atmosphere and Ocean Research Institute[END_REF]: CO 2 was injected into a quartz tube together with 4 mg of iron powder prereduced at 450 °C and graphitized by heating at 630 °C for 6 h. The samples were measured using a single-stage accelerator mass spectrometer at the University of Tokyo Atmosphere and Ocean Research Institute [START_REF] Yokoyama | A single stage Accelerator Mass Spectrometry at the Atmosphere and Ocean Research Institute, The University of Tokyo[END_REF].

Decalcified, dried samples were placed in pre-combusted glass cups. The samples were further graphitized according to the modified methods of [START_REF] Yokoyama | Developing ultra smallscale radiocarbon sample measurement at the University of Tokyo[END_REF]. Briefly, dried samples (3.14 to 13.35 mg, typically 10 mg) were combusted in an evacuated quartz tube with copper oxide at 500°C for 30 min and at 850°C for 2 h. The CO2 gas was cryogenically purified in a vacuum line and reduced to graphite with hydrogen and an iron catalyst at 550°C for 10 h. We measured the Δ 14 C values with an accelerator mass spectrometer (AMS) at the Atmosphere and Ocean Research Institute [START_REF] Yokoyama | A single stage Accelerator Mass Spectrometry at the Atmosphere and Ocean Research Institute, The University of Tokyo[END_REF].

The Δ 14 C (‰) value was defined as follows [START_REF] Stuiver | Discussion: reporting of 14C data[END_REF]: δ 14 C (‰) = δ 14 C -2(δ 13 C + 25)(1 + δ 14 C /1000). The Δ 14 C value of the international standard (oxalic acid) takes into account radioactive decay since 1950 [START_REF] Stuiver | Discussion: reporting of 14C data[END_REF]. The 1 sigma analytical precision of the Δ 14 C measurements was within 5‰, typically within 3‰ [START_REF] Yokoyama | Efficient radiocarbon measurements on marine and terrestrial samples with single stage Accelerator Mass Spectrometry at the Atmosphere and Ocean Research Institute[END_REF]. Cross contamination amongst samples were avoided after careful treatment of samples as well as preparations [START_REF] Yamane | Small-to ultra-small-scale radiocarbon measurements using newly installed single-stage AMS at the University of Tokyo[END_REF]. Rigorous background assessments was conducted to ensure reliability of results [START_REF] Yokoyama | Developing ultra smallscale radiocarbon sample measurement at the University of Tokyo[END_REF], Yamane et al. 2019).

2.5) Data analyses

The nonparametric Kruskal-Wallis test was used to examine differences in isotopic composition (δ 13 C, δ 15 N and Δ 14 C) of consumers. All statistical analyses were performed using R. Values are presented as means with standard deviations (SD).

Bayesian isotopic mixing models were employed to determine contributions of bacteria to diets of consumers. SIAR (Version 4.2; [START_REF] Parnell | Source partitioning using stable isotopes: coping with too much variation[END_REF])) incorporates the variability of consumer and trophic enrichment factors (TEFs, i.e. the net isotopic composition change in a consumer and its ingested food sources) to produce the percent contribution of each source to a consumer's diet. TEFs are key factors when it comes to evaluate contributions of food sources to animal diets. TEFs used were 1.1 ± 0.5‰ for δ 13 C, 2.2 ± 0.5‰ for δ 15 N and 2.0 ± 0.7‰ for δ 34 S [START_REF] Mccutchan | Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur[END_REF]. These TEFs are typically used in isotope studies and are appropriate when consumers are not starved [START_REF] Vandenberg | Intracellular pH recovery during respiratory acidosis in perfused hearts[END_REF][START_REF] Lefebvre | The stony road to understand isotopic enrichment and turnover rates: insight into the metabolic part[END_REF]. Variations of 20% in these TEFs do not change the conclusion of the present study as they induce only small variations of 2.3 ± 1.9% in model results.

A 2-step procedure was performed to run the SIAR modelling. First, TEFs [START_REF] Mccutchan | Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur[END_REF] were subtracted from isotopic compositions of consumers caught in control bay in order to determine the isotopic compositions of bulk diets of each consumer in an environment without bacteria. Average values and standard deviations of those results were then used as 'food sources' in models using isotopic compositions of consumers from Bouillante bay in contact with bacteria. For each consumer, 2 potential food sources were considered: i) chemoautotrophic bacteria and ii) average isotopic composition of consumers' bulk food sources in control Bay and three different models were run with i) 13 C and 15 N, ii) 14 C and iii) 13 C, 15 N and 14 C. This evaluation of the contribution of bacteria to diets is based on the assumption that bacteria are the only food item whose contribution to faunal diets changes between Bouillante and control bays.

The model was run with 106 iterations and burn-in size was set as 105. Model solutions are presented using credibility intervals of probability density function distributions [START_REF] Parnell | Source partitioning using stable isotopes: coping with too much variation[END_REF].

3) Results

3.1) Stable isotopes (δ 13 C and δ 15 N)

Bacteria released by the geothermal plant of Bouillante presented a δ 13 C of -17.3 ± 1.5 ‰ and δ 15 N of -4.7 ± 1 ‰. Among potential consumers from control site, highest δ 15 N was found in the three fish species: S. partitus (δ 15 N = 7.9 ± 0.2), A. saxatilis (δ 15 N = 7.7 ± 0.4) and A. bahianus (δ 15 N = 6.8 ± 0.5), fireworm H. carunculata (δ 15 N = 6.5 ± 0.5) and the sea urchin D. antillarum (δ 15 N = 6.4 ± 0.2). Except for A. saxatilis (δ 13 C = -16.3 ± 0.5), all those consumers from control site also present highest δ 13 C: S. partitus (δ 13 C = -15.1 ± 0.5), A. bahianus (δ 13 C = -15.6 ± 0.7), H. carunculata (δ 13 C = -14.4 ± 1.2) and D. antillarum (δ 13 C = -15.1 ± 0.5) (Fig. 2).

Between the

3.2) Radiocarbon (Δ 14 C)

Water released by the geothermal plant is composed by pumped geothermal water mixed with surrounding sea water from the bay and presented a Dissolved Inorganic Carbon (DIC) Δ 14 C of -682 ‰ (n = 1) whereas the water from the control site presented a mean DIC Δ 14 C of 25 ± 2 ‰ (n = 2). Sulfur bacteria released by the geothermal plant of Bouillante through the discharge channel presented a mean Δ 14 C of -159 ± 32 ‰ (n = 2). Among potential consumers from control site, highest Δ 14 C was found in A. bahianus (35.5 ± 2.0) whereas the lowest was found in A. fistularis (25.0 ± 1.1) (Fig. 2). Between the Bouillante and control station, all studied organisms presented a significantly lower Δ 14 C except Balanus sp., Pseudopterogorgia sp., A. fistularis and I. birotulata (Kruskal-Wallis tests, p > 0.05). Most important decrease in Δ 14 C were observed for A. saxatilis, A. bahianus and D. antillarum and S. partitus (Fig. 3).

3.3) Model SIAR

SIAR model simulations using δ 13 C-δ 15 N, Δ 14 C and combined 13 C-δ 15 N-Δ 14 C predict different estimates of chemoautotrophic bacteria contributions to studied organisms (Fig. 4).

Mean contribution of bacteria in diet (%) were higher with models using δ 13 C-δ 15 N versus models using Δ 14 C for urchin and fish: D. antillarum (27 vs. 44), A. bahianus (47 vs. 54), A. saxatilis (37 vs. 63) and S. partitus (18 vs. 32) (Fig 5). Mean contribution of bacteria in diet (%) were similar between both models for A. fistularis (10 vs. 12) and lower with models using δ 13 C-δ 15 N versus models using Δ 14 C for all other organisms: H. caruncalata (32 vs. 13), S. tenuis (34 vs. 7), Balanus sp. (45 vs. 14), Pseudopterogorgia sp. (13 vs. 3), and I. birotulata (13 vs. 2) (Fig. 5). Mean bacterial contributions (%) estimated with SIAR models using combined 13 C-δ 15 N-Δ 14 C values (with lower and higher 95% credibility intervals) to the diets of D. antillarum, A. bahianus,A. saxatilis,S. partitus,H. caruncalata,Balanus sp.,Pseudopterogorgia sp.,[21][22][23][8][9][10][11][12], 15 (0-27), 4 (0-14), 5 (0-15) and 2 (0-7) (Fig. 4).

Mean values of the interval between lower and higher 95% credibility interval for all studied organisms was 40 ± 22% for δ 13 C-δ 15 N models, 39 ± 24% for Δ 14 C models and 24 ± 12% for 13 C-δ 15 N-Δ 14 C models but those values were not significantly different (Kruskal-Wallis tests, NS).

4) Discussion

4.1) Radiocarbon as a powerful tracer to understand trophic relationship

The use of radiocarbon to evaluate the trophic role of a food resource necessitates a specific Δ 14 C composition of this food source compared to other items available.

Chemoautotrophic microbes using 14 C depleted carbon releases by volcanic activity (CO2 or CH4) exhibit depleted 14 C concentrations compared to atmospheric carbon [START_REF] Bruns | Regional sources of volcanic carbon dioxide and their influence on 14 C content of present-day plant material[END_REF][START_REF] Williams | Dietary carbon sources of mussels and tubeworms from Galapagos hydrothermal vents determined from tissue 14 C activity[END_REF]. Bouillante Bay is an appropriate study site as the source of geothermal bacteria is artificial and consequently restricted spatially. Opportunist species such as fish and urchin ingest bacteria from geothermal water as it has been shown using C, N and S stable isotopes [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF]) and heavy metal tracers (Ruiz et al. In prep). The present study reveals a specific radiocarbon content of geothermal bacteria changing the radiocarbon composition of their consumers in Bouillante Bay confirming the utility of this tracer in food web ecology.

In order to evaluate the potential advantages of radiocarbon, we compared Δ 14 C data and stable isotope compositions (δ 13 C and δ 15 N) simultaneously measured in Bouillante and Control stations. Similar analyses in a control station located 400 m away from the discharge channel were realized in a previous study and revealed analogous results concerning δ 13 C of urchin, fireworm and sponge [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF]. Results were also similar for δ 15 N with highest values for fish, urchin and polychaeta confirming the higher trophic levels of those species. Similar results indicate a relatively stable structure of the food web allowing comparison between study despite the different sampling period. Differences in isotopic composition of potential consumers between stations from control and close to geothermal plant were not totally similar according to studies. None δ 13 C composition was different in the present study, whereas a lower δ 13 C was previously observed close to the geothermal plant for two species (D. antillarum and A. saxatilis) [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF]. Contrariwise all species from the station close to the plant present a lower δ 15 N in the present study whereas this difference was only observed for three species previously (D. antillarum, A. saxatilis and S. partitus). In both studies, average isotopic compositions of potential consumers from control stations were used to evaluate contribution of geothermal bacteria to the diet of consumers found close to the release channel. The present model using δ 13 C and δ 15 N suggest an ingestion of bacteria by all consumers including filter feeders and omnivorous predators whereas importance of bacteria was previously shown to be limited in their diets using stable isotope [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF].

Water sample dissolved inorganic carbon (DIC) and fauna from the control site present a Δ 14 C fluctuating between 25 and 35 ‰ reflecting atmospheric CO2. Stream water can contain DIC from weathered carbonates and old organic matter with lower Δ 14 C than contemporary atmospheric CO2 [START_REF] Fellman | Evidence for the assimilation of ancient glacier organic carbon in a proglacial stream food web[END_REF]. In the control site, the influence of freshwater discharge would be limited due to its estrangement with river mouths and since rivers of Basse Terre do not contain carbonate bedrock due to the volcanic origin of the island. Several species from Bouillante present similar Δ 14 C composition suggesting that the bias due to freshwater contribution can be reasonably set apart.

The DIC of water released by the plant present a Δ 14 C of -682‰. The geothermal water pumped by the plant is diluted with seawater from the bay in order to reach a temperature lower than 45°C before its release. Δ 14 C of the DIC of geothermal water would consequently be lower than -682‰ with a composition characteristic of DIC from magmatic activity (Δ 14 C = -1000‰ or "dead carbon") [START_REF] Corliss | Submarine thermal springs on the Galapagos rift[END_REF]. Bacteria are synthetizing organic matter in their cells by using carbon available in their surrounding environment [START_REF] Hansman | The radiocarbon signature of microorganims in the mesopelagic ocean[END_REF][START_REF] Nishida | Novel reverse radioisotope labelling experiment reveals carbon assimilation of marine calcifiers under ocean acidification conditions[END_REF]. Bacteria covering pebbles of the discharge channel should consequently present a Δ 14 C similar to Δ 14 C of DIC of water release by the plant. However, a difference of -159 versus -682 was observed between carbon of bacteria and water respectively. Bacteria would integrate carbon over a long time period and the punctual measurement done in water in this study is not necessarily representative of water composition at longer time scale. Despite this discrepancy, geothermal bacteria present a distinct radiocarbon composition compared to atmospheric CO2 allowing the use of radiocarbon as a trophic tracer.

4.2) Ecological relations revealed from SIAR model

SIAR model using radiocarbon data quantify the role of geothermal bacteria in the diet of each organism. Results are similar to previous SIAR modelling using δ 13 C, δ 15 N and δ 34 S stable isotopes [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF]): fish and urchin ingest more bacteria than others organisms. The 3 mobile fish ingest bacteria as it was previously shown with i) SIAR modelling, ii) evolution of isotopic composition of fish after the temporal stop of bacterial production, iii) higher abundance of fish when the plant was active [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF] and Hg composition of liver and muscle of each fish species (Ruiz et al. In prep). For the urchin D. antillarum, sulfur bacteria represent an important part of the diet (32%) but lower than previously modelled (66%). This difference can be due to the mobility of this species and its ability to selectively ingest food [START_REF] Tuya | Food preference of the sea urchin Diadema antillarum in Gran Canaria (Canary Islands, central-east Atlantic Ocean)[END_REF], Cabanillas-Téran et al. 2019) changing diet composition between each study. The opportunist omnivorous scavenger fireworm Hermodice carunculata [START_REF] Jumars | Diet of worms emended: an update of polychaete feeding guilds[END_REF] does not ingest important amount of sulfur bacteria as previously shown [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF]. All other grazers are filter feeders with different strategy to extract their food from a dilute environment [START_REF] Riigsård | Particle capture mechanisms in suspension-feeding invertebrates[END_REF].

None of them ingest significant amount of geothermal bacteria as previously shown in Bouillante for passive suspension-feeder [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF] with the gorgonian Pseudopterogorgia sp. and active suspension-feeders such as bivalves (S. tenuis using gills and labial palps), barnacle (Balanus sp. using cirri feeding legs) and sponges (I. birotulata and A. fistularis pumping water to choanocyte chambers where particles are retained). A. fistularis is a bacteriosponge with a diet relaying on large amount of symbiotic bacteria using dissolved carbon resource [START_REF] Reiswig | Partial carbon and energy budgets of the bacteriosponge Verongia fistularis (Porifera, Demospongiae) in Barbados[END_REF][START_REF] Hentschel | Marine sponges as microbial fermenters[END_REF]. Radiocarbon does not allow to detect the influence of geothermal water in A. fistularis suggesting that the plant would have a limited influence on the total amount of dissolved carbon. The vent mussel Bathymodiulus thermophiles host chemosymbiotic bacteria giving the mussel the ability to perform CO2 fixation [START_REF] Ponnudurai | Genome sequence of the sulfur-oxidizing Bathymodiolus thermophilus gill endosymbiont[END_REF]. Due to the fixation of volcanic DIC, B. thermophiles present a depleted Δ 14 C close to deep sea vent whereas 8 m away from this vent the influence of geothermal DIC was not detectable anymore in mussel [START_REF] Williams | Dietary carbon sources of mussels and tubeworms from Galapagos hydrothermal vents determined from tissue 14 C activity[END_REF] suggesting a rapid dilution of dissolved carbon from geothermic activity at decimeter to meter scale [START_REF] Nomaki | Nutritional sources of meio-and macrofauna at hydrothermal vents and adjacent areas: natural-abundance radiocarbon and stable isotope analyses[END_REF]. Similar process can occur in the present study where all biological samples were collected as close as possible to the water discharge outlet with a distance lower than 5 m (according to the availability of species). However, this distance may be too large to detect influence of DIC in sponge at really small spatial scale due to rapid DIC dilution.

Mixing model using simultaneously δ 13 C, δ 15 N and Δ 14 C suggest an ingestion of bacteria limited to opportunist and moving fauna (fish and urchin) whereas fixed filter feeders were unaffected as revealed by a previous study in Bouillante [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF]. Similar restriction of the trophic role of geothermal bacteria according to feeding mode has been observed in shallow vent of California [START_REF] Trager | Chemoautotrophic sulphur bacteria as food source for mollusks at intertidal hydrothermal vents: evidence from stable isotopes[END_REF].

Food web of vents in deep sea are mainly supported by geothermal bacteria [START_REF] Lonsdale | Clustering of suspension-feeding macrobenthos near abyssal hydrothermal vents at oceanic spreading centres[END_REF] whereas in shallow vent, the energy is supplied by both chemosynthesis and photosynthesis [START_REF] Tarasov | Deep-sea and shallow-water hydrothermal vent communities: two different phenomena?[END_REF]. In intertidal vent of California, geothermal bacteria are ingested by grazers and not by filter feeders (bivalves) as in Bouillante Bay [START_REF] Trager | Chemoautotrophic sulphur bacteria as food source for mollusks at intertidal hydrothermal vents: evidence from stable isotopes[END_REF]. In Italy, most organisms ingest bacteria including filter feeders such as bivalves but not sponges [START_REF] Southward | On the biology of submarine caves with sulphur springs: 13 C/ 12 C ratios as a guide to trophic relations[END_REF][START_REF] Southward | Behaviour and feeding of the nassariid gastropod Cyclope neritea, abundant at hydrothermal brine seeps off Milos (Aegean sea)[END_REF]. In Taiwan, grazers, predators and filter feeders represented by sea anemone, slipper limpet and coral ingest bacteria even if they do not represent the majority of their diet [START_REF] Wang | Autochtony and isotopic niches of benthic fauna at shallow-water hydrothermal vents[END_REF].

δ 13 C and δ 15 N values are routinely used to track organic matter moving through food web [START_REF] Peterson | Stable isotopes in ecosystem studies[END_REF]). In the present study, the simultaneous measurement of those classical isotope and radiocarbon leads to results slightly different. Compared to modelled diet using radiocarbon, the use of δ 13 C and δ 15 N data lead to underestimate the role of geothermal bacteria for diadema urchin and 3 fish species whereas this role is overestimated for all other studied organisms. Radiocarbon leads to results closer to result previously modelled [START_REF] Pascal | Influences of geothermal sulfur bacteria on a tropical coastal food web[END_REF]. Most probable explanation for this difference would be due to trophic discrimination as δ 13 C and δ 15 N are affected by metabolic activity during the production of organic matter [START_REF] Laws | Effect of growth rate and CO 2 concentration on carbon isotope fractionation by the marine diatom Phaedactylum tricornutum[END_REF]). Uncertainty associated with factor of discrimination can results in error higher than 30% of source contribution estimated in mixing model [START_REF] Vander Zanden | Variations in δ 15 N and δ 13 C trophic fractionation: implications for aquatic food web studies[END_REF][START_REF] Medina-Contreras | Trophic structure of fish communities in mangrove systems subject to different levels of anthropogenic intervention, Tropical Eastern Pacific, Colombia[END_REF]). On the contrary radiocarbon are not affected by fractionation associated with trophic level, productivity and composition of the prey. Potential biases are reduced and radiocarbon consequently constitute a more robust tracer of carbon [START_REF] Ishikawa | Natural C-14 provides new data for stream food-web studies: a comparison with C-13 in multiple stream habitats[END_REF]) more sensitive in detecting supplementary feeding events [START_REF] Eisenmann | Radiocarbon as a novel tracer of extra-antarctic feeding in southern hemisphere humpback whales[END_REF].

Radiocarbon analyses are more expensive than stable isotopes ones but it becomes gradually i) more affordable and ii) less demanding about sample biomass. As a result radiocarbon will probably be more used in food web study in coming decade [START_REF] Larsen | Radiocarbon in ecology: Insights and perspectives from aquatic and terrestrial studies[END_REF]. The utility of radiocarbon is limited to food web containing a food item with a specific depleted value compared to others. Such depletion can be due to food items from geothermal activity but also in food items from the decay of old organic matter such as hydrocarbon and methane. In such situation, the present study confirms the utility of this tracer giving more reliable result than the classical use of stable isotope (δ 13 C and δ 15 N).

  Bouillante and control stations, studied organisms do not present a significantly different δ 13 C values (Kruskal-Wallis tests, p > 0.05) except S. partitus and I. birotulata (Fig 2). In Bouillante, all studied organisms present significantly lower δ 15 N than in the control station (Kruskal-Wallis tests, p > 0.05) (Fig 2).

FigureFigure 1

 1 Figure

Figure 4 .

 4 Figure 4. Contribution (% by weight) of bacteria to the diet of 1) D. antillarum, 2) A.

Figure 5 .

 5 Figure 5. Contribution (% by weight) of bacteria to the diet of each organism evaluated using

Acknowledgements 444

We thank Sébastien Cordonnier for his assistance in the field. 445


Xavier Hubert Jaglin

Karine Poirier

Yoann Saillour

Emmanuelle Buhler

Guoling Tian

Nadia Bahi-Buisson

Catherine Fallet-Bianco

Fran Oise Phan-Dinh-Tuy Ç 1 #

Xiang Peng Kong

Pascale Bomont

La Titia Castelnau-Ptakhine

Sylvie Odent

Philippe Loget

Manoelle Kossorotoff

Irina Snoeck

Ghislaine Plessis

Philippe Parent

Cherif Beldjord

Carlos Cardoso

Alfonso Represa

Jonathan Flint

David Anthony Keays

Nicholas Justin Cowan
email: <nicholas.cowan@med.nyu.edu

Jamel Chelly

Juliana Kinderziekenhuis Polikliniek

Mutations in the beta-tubulin gene TUBB2B result in asymmetrical polymicrogyria

Polymicrogyria is a relatively common but poorly understood defect of cortical development characterized by numerous small gyri and a thick disorganized cortical plate lacking normal lamination. We show an association between bilateral asymmetrical polymicrogyria and mutations in a -tubulin gene, , in four patients and a 27 GW (gestational week) fetus. de novo β

TUBB2B

Neuropathological examination of the fetus revealed an absence of cortical lamination associated with the presence of ectopic neuronal cells in the white matter, and in the leptomeningeal spaces due to breaches in the pial basement membrane. In utero RNAi-based inactivation demonstrates that is required for neuronal migration. We also show that two disease-associated TUBB2B mutations lead to an impaired formation of tubulin heterodimers. These observations, together with previous data, demonstrate that disruption of microtubule-based processes underlies a large spectrum of neuronal migration disorders that includes not only lissencephaly/pachygyria, but also polymicrogyria malformations.

The crucial role of the tubulin superfamily in diverse cellular processes and the association of mutations with a broad [START_REF] Dutcher | The tubulin fraternity: alpha to eta[END_REF] TUBA1A lissencephaly spectrum -led us to hypothesize that mutations in other tubulin genes that are highly expressed during CNS [2 4 ] development might also result in malformations of cortical development. In a previous screen of agyria/pachygyria patients, we excluded the implication of , and . In this study we report the screening of 3 additional candidate tubulin genes ( TUBA1B TUBA1C

TUBB3 [START_REF] Poirier | Large spectrum of lissencephaly and pachygyria phenotypes resulting from de novo missense mutations in tubulin alpha 1A(TUBA1A)[END_REF] ) in patients with a wide range of cortical dysgeneses, including polymicrogyria (PMG) syndromes associated with epilepsy TUBB2A,B,C and/or neurodevelopmental delay (see Material and Methods section). Although no non-synonymous variations were found either in or in (see alignment in ), heterozygous missense mutations were found in ( ) in TUBB2A TUBB2C Supplementary Fig. 1 TUBB2B Fig. 1a four unrelated individuals and one fetus. (p.F265L) and c.935C T (p.T312M) affect residues that are rigidly conserved from yeast to human and reside in > Supplementary Fig. 2 exon 4 ( , ). Consistent with a origin of the mutations, none were found in the parents of affected Fig. 1a Table 1 de novo TUBB2B individuals or in 360 normal controls (see referenced polymorphisms in ). Brain MRI sequences revealed that all Supplementary Fig. 1 patients share the presence of a complex brain dysgenesis with bilateral, asymmetrical, and anteriorly predominant polymicrogyria (PMG), fusion of the caudate and putamen with internal capsule hypoplasia, corpus callosum agenesis or dysgenesis and, in most cases, cerebellar and pons atrophy ( , , ). In addition, neurohistopathological analysis of the fetal brain showed Fig. 1b-j Table 1 Supplementary Fig. 3 asymmetrical bilateral PMG with the absence of a corpus callosum, several nodular cluster of ectopic neurons in both hemispheres and a disorganization of cortical layering (

) including the presence of radial columnar heterotopic neurons in the white matter in the two Fig. 1k-r hemispheres ( ). These abnormalities strongly suggest migration defects and perturbations of axon tract formation associated with Fig. 1m mutations in . In addition to the typical unlayered polymicrogyric cortex, the analysis of the left hemisphere reveals TUBB2B overmigration of MAP2 neurons through breaches in the pial basement membrane (BM) ( and ). Analysis + Fig. 1l,n,r Supplementary Fig. 4 of radial glial fibers revealed a striking disorganization beyond the pial BM ( ). These observations recall the phenotype of mice Fig. 1p inactivated for , a gene associated with bilateral frontoparietal PMG in humans . The macroscopic aspect and folding of the Gpr56 [5 ] [6 ] cerebellum appeared similar to the cerebellum of a control fetus of 27 GW. At the microscopic level, many nodular heterotopia were observed in both cerebellar hemispheres.

To investigate the association between mutations and neuronal migration disorders, we studied the expression of TUBB2B Tubb2b

during mouse brain development by hybridization and qRT-PCR and analyzed the consequences on the cortical neuronal migration in situ of loss of function induced by in utero RNA interference. TUBB2B

Sections of embryos at embryonic day 14.5 (E14.5) and 16.5 (E16.5) showed strong labeling restricted to central and peripheral nervous systems (See ). Using sagittal sections of the brain at E16.5, we found that expression predominated in the Supplementary Fig. 5 cortical plate and also within a thin layer in the subplate, whereas no signal was observed in the marginal zone and fainter labeling was seen in the ventricular and intermediate zones (Supplementary ). The strong labelling in the developing cortex Supplementary Fig. 5a-e subsequently decreases after birth although labeling remains intense in the adult cerebellum, hippocampus and olfactory bulb ( Tubb2b

). Taken together these data suggest that is strongly expressed in postmitotic neurons with dominant Supplementary Fig. 5 Tubb2b expression during neuronal migration and differentiation -, and to a lesser extent in progenitor cells. RT-PCR analysis also showed [7 9 ] that is expressed in astro-glial cells and C6 glial cells (data not shown). Tubb2b

We used rat RNAi approach to knock-down expression by about 50 % at embryonic day 15.5 (E15.5), a time in utero [START_REF] Bai | RNAi reveals doublecortin is required for radial migration in rat neocortex[END_REF] Tubb2b coincident with the migration of neurons within the cortex, to mimic the consequence of heterozygous loss of function mutations. We tested different small hairpin RNAs (shRNA) and used two shRNAs targeting either the coding sequence (CDS-sh) or the 3 untranslated ′ region (3 UTR-sh) that repressed expression in vitro by approximately 60%, while scrambled controls did not destabilize the ′ Tubb2b mRNA ( ). These shRNAs combined with a Red Fluorescent Protein (RFP)-encoding reporter construct Tubb2b Supplementary Fig. 6a-b were electroporated into progenitor cells located of the ventricular zone (VZ) of E15 rat neocortices. These cells give rise to further young neurons expressing the fluorescent protein as they migrate towards the cortical plate. In E20 brain sections, we observed that neurons electroporated 5 days previously with RFP alone reached the cortical plate as expected ( ). However in utero expression of 3 Fig. 2a-b ′ UTR-sh induced a significant arrest of cells within the sub-ventricular zone (SVZ)/intermediate zone (IZ) ( ; stratum 4: Fig. 2c-d,j-k F(3,42)=21.716, p 0.0001; stratum 5:F(3,42)=20.394, p 0.0001). To further validate the specificity of our results, we performed

< < in utero
RNAi with the coding sequence CDS-sh and found that it leads to the same migration arrest ( , ). We also Fig. 2e Supplementary Fig. 7 showed that the expression of both scrambled-sh controls did not disrupt migration (

). Finally, we performed a rescue experiment Fig. 2f,g in which we cotransfected a bicistronic construct driving the expression of and Green Fluorescent Protein (GFP) as a reporter. Tubb2b

Although the expression of this construct alone does not alter migration ( ), it significantly suppresses the blocking effect of Fig. 2h 3'UTR-sh on migration ( ; strata 8-10: F(3,42)>23.4,p 0.0001). This RNAi-based approach reinforces the evidence that Fig. 2i,k < microtubules act as a critical node during corticogenesis and strongly implicates in neuronal migration. Tubb2b/TUBB2B

To define the functional consequences of the mutations in , we examined the potential implications of each of the mutated TUBB2B residues on the known structure of the -tubulin polypeptide , ( ). S172 resides in a loop that forms part of the guanosine β [11 12 ] Fig. 3a nucleotide-binding site, which when mutated to a proline (S172P) is predicted to disrupt a hydrogen bond and to destabilize the GTP pocket. L228 and F265 are either in the vicinity of or part of the GTP/GDP binding site, with potential consequences for GTP binding. The remaining mutations appear to be less consequential in terms of the GTP binding and overall stability of the protein. Because they are located on the surface, they may interfere with specific partner interactions.

We then investigated the ability of the -tubulin mutants to produce functional / tubulin heterodimers through the complex

β α β
chaperone-dependent folding pathway , ( ) by transcription/translation in rabbit reticulocyte lysate . All mutant proteins [13 14 ] Fig. 3b 15

were translated as efficiently as a wild type control ( ). In contrast, analysis under native conditions revealed a range of reaction Fig. 3c products ( ) that could be assigned on the basis of their electrophoretic mobilities , . In the case of TUBB2B mutant Fig. 3d,e [14 16 ] proteins, these products frequently differed both quantitatively and qualitatively from the wild type control (See ). Supplementary Table 1 Most conspicuously, two mutant proteins (p.F265L and p.S172P) completely failed to yield the intermediate corresponding to Tubulinspecific Chaperone A (TBCA)/ -tubulin (

). In addition, the yield of native / heterodimers produced following a chase β Fig. 3d,e α β with added native tubulin was either slightly reduced (in the case of p.I210T and p.T312M), dramatically reduced (in the case of p.L228P and p.F265L), or undetectable (in the case of p.S172P) ( ). (For kinetic analyses, see also and Fig. 3e Supplementary Results

). Supplementary Fig. 8 We then investigated the ability of the -tubulin mutants to produce functional / tubulin heterodimers through the complex

β α β
chaperone-dependent folding pathway , ( ) by transcription/translation in rabbit reticulocyte lysate . All mutant proteins [13 14 ] Fig. 3b 15

were translated as efficiently as a wild type control ( ). In contrast, analysis under native conditions revealed a range of reaction Fig. 3c products (

) that could be assigned on the basis of their electrophoretic mobilities , . In the case of TUBB2B mutant Fig. 3d,e [14 16 ] proteins, these products frequently differed both quantitatively and qualitatively from the wild type control (See ). Supplementary Table 1 Most conspicuously, two mutant proteins (p.F265L and p.S172P) completely failed to yield the intermediate corresponding to Tubulinspecific Chaperone A (TBCA)/ -tubulin (

). In addition, the yield of native / heterodimers produced following a chase β Fig. 3d,e α β with added native tubulin was either slightly reduced (in the case of p.I210T and p.T312M), dramatically reduced (in the case of p.L228P and p.F265L), or undetectable (in the case of p.S172P) ( ). (For kinetic analyses, see also and Fig. 3e Supplementary Results

. Supplementary Fig. 8 ]

To examine the mechanism of defective heterodimer assembly of the p.F265L and p.S172P mutant polypeptides in detail, we did reconstituted folding reactions using purified components , . We observed the generation of TBCD/ -tubulin co-complexes in vitro [14 16 ] β in reactions performed with p.F265L and p.S172P ( ) that might be ascribable to the relatively high concentrations of CCT and Fig. 4a TBCD in these reactions compared to the more physiological concentrations in reticulocyte lysate. Indeed, when the abundance of TBCD was reduced by a factor of 5 in reactions performed with a constant level of CCT, the yield of the wild type -tubulin/TBCD co-complex β diminished to 25 of the original level, and the F265L -tubulin/TBCD co-complex was reduced to an undetectable level (

).

% β

Fig. 4c,d Similar data were obtained for the p.S172P mutation . We conclude that in addition to a dramatic failure of [Supplementary Fig. 9 ] p.F265L and p.S172P CCT-generated folding intermediates to stably interact with TBCA, these mutations also result in a reduced efficiency of intermediate interaction with TBCD (See also . To assess the competence of mutant heterodimers Supplementary Results ] expressed by transcription/translation to incorporate into microtubules, we analyzed their ability to co-cycle with native brain microtubules and to co-assemble with microtubules upon transfection into cultured cells. In the cases of p.F265L and p.S172P we observed 1) a in vitro very low yield of labeled heterodimers incorporated into microtubules and 2) a further diminution between the first (1) and second [START_REF] Keays | Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans[END_REF] cycles of polymerization/depolymerization, suggesting significant instability (

). Upon heterologous overexpression by transfection Fig. 5a in cultured cells, we found that 3 mutants (p.I210T, p.L228P, p.T312M) behaved indistinguishably from the wild-type protein in that they were efficiently incorporated into interphase microtubules (data not shown). In contrast, in the case of the p.S172P and p.F265L mutants, there was scant evidence of incorporation into well-defined microtubules ( ) (See also for assessment of Fig. 5b Supplementary Results microtubule dynamics). Taken together, these experiments show that p.S172P and p.F265L are significantly compromised in their ability to properly assemble into microtubules , and are consistent with our in vitro transcription/translation experiments in which neither in vivo of these mutant proteins yielded significant amounts of polymerization competent heterodimers ( , ). Fig. 3e 5a We further sought to test whether expression of the p.S172P and unrelated p.T312M mutants could complement the phenotype in vivo caused by knockdown of . We electroporated the pCAGIG-p.S172P(or p.T312M)--IRES-GFP construct either alone or in Tubb2b Tubb2b

combination with the 3 UTR-sh and analyzed the position of electroporated cells within the cortex. We found that, although expression of ′ each mutant alone does not massively affect migration ( ), expression of each mutant in the knock-down context maintains the Fig. 5e,g cells stalled within the SVZ/IZ ( ) and fails to complement the RNAi effect. Fig. 5f,h In this study, we implicate mutations in as causative of brain malformations encompassing asymmetrical PMG associated TUBB2B with an unlayered cortex, heterotopic neuronal cells in the white matter and neuronal overmigration through the pial BM. It is worthwhile mentioning that corpus callosum dysgenesis, dysmorphic basal ganglia, cerebellum dysplasia and brainstem hypoplasia are common features to -related agyria/pachygyria and -related PMG. Mutations in and genes are also known to be TUBA1A TUBB2B GPR56 SRPX2 associated with PMG. However the diagnosis of -related PMG could be evoked through assessment of clinical and imaging TUBB2B criteria highlighted in . Supplementary Table 2 In this study, we implicate mutations in as causative of brain malformations encompassing asymmetrical PMG associated TUBB2B with an unlayered cortex, heterotopic neuronal cells in the white matter and neuronal overmigration through the pial BM. It is worthwhile mentioning that corpus callosum dysgenesis, dysmorphic basal ganglia, cerebellum dysplasia and brainstem hypoplasia are common features to -related agyria/pachygyria and -related PMG. Mutations in and genes are also known to be TUBA1A TUBB2B GPR56 SRPX2 associated with PMG. However the diagnosis of -related PMG could be evoked through assessment of clinical and imaging TUBB2B criteria highlighted in . Supplementary Table 2 Our data show that the five newly discovered disease-associated mutations involve a spectrum of tubulin in vitro TUBB2B

heterodimer assembly defects (summarized in ) leading to loss of function in the cases of p.S172P and p.F265L. Supplementary Table 1 The two most severe defects observed in vitro (p.S172P and p.F265L) are associated with the most and the least severe clinical phenotypes, respectively, suggesting that there is no simple correlation in the panel of 5 mutated patients. We also establish that loss of function is associated with defective migration and mislocalization of developing neurons within the cortex, suggesting that in vivo -related forms of PMG are primarily due to haploinsufficiency, though dominant negative effect can not be excluded for the TUBB2B p.S172P mutation. For those mutations that have less or no apparent impact on tubulin heterodimer assembly, the functional defect seems likely to concern a subtle effect on either microtubule dynamics, or on the interaction with one or more microtubule interacting proteins (MAPs) that are critical for proper cortical neuronal migration, or both. The cellular consequences of mutations, and the TUBB2B mechanisms by which this results in polymicrogyria, an unlayered cortex, and heterotopic neuronal cells remain unknown. We propose that the neuropathophysiology of -related PMG might result from a combination of both neuronal migration impairment and TUBB2B radial glia dysfunction that lead respectively to ectopic neurons in the white matter and cerebellum, and to pial membrane breaches (see ).

Supplementary Discussion

In higher eukaryotes, -and -tubulins are encoded by a multigene family that are evolutionary conserved among different species α β [START_REF] Little | Comparative analysis of tubulin sequences[END_REF] and are differentially expressed , . To explain the need for these highly conserved multiple genes, it has been hypothesized that the [18 19 ] different isotypes may be required to form specific sets of microtubules that carry out unique functions18. Though this hypothesis is still a matter of debate, our data showing that mutations in and are associated with different gyral abnormalities argue in TUBA1A TUBB2B

favor of specific roles of and during corticogenesis and neuronal migration (see ). It is also TUBB2B TUBA1A Supplementary Table 2 possible that subtle differences of spatio-temporal profiles of and expression (i.e. populations of interneurons, radial TUBA1A TUBB2B glia cells and astro-glial cells) may account for some of the cortical phenotypic differences.

METHODS

Patients and Analysis of TUBB2B

Patients' DNA or blood samples and informed consent (from all patients' parents) were obtained according to the guidelines of local institutional review boards at Cochin Hospital and INSERM (French National Institute of Health and Medical Research). was TUBB2B screened in a total of 168 sporadic cases selected with clinical and brain imaging features compatible with a diagnosis of either lissencephaly (n=120) or polymicrogyria syndromes (n=48). Patients with lissencephaly included those with agyria/pachygyria (n=105) or subcortical laminar heterotopia (n=15) with (n=13) or without (n=107) cerebellar hypoplasia or dysplasia. In addition to this cohort of living patients, we also screened in 5 fetuses (using DNA samples derived from skin tissue), which had been sent to our TUBB2B diagnostic center after medical abortions and neuropathological analyses (See details in section). Supplementary Methods

Neuropathological procedures

Neuropathological analyses were performed on 5 fetuses (aged from 23 to 35 gestational weeks) in accordance with French law.

Briefly, after removal from the skull, each brain was fixed in 10 (v/v) formaldehyde solution containing NaCl (9 g/l) and ZnSO (3 g/l) % 4 for a variable time (depending on the volume of the brain) from 3 to 6 weeks. Brains were cut in a coronal plane and sections involving one or both hemispheres were embedded in paraffin. Paraffin blocks were sectioned into either 5 m (brainstem and cerebellum) or 8 10 μ -μ m (hemispheres) thick sections and stained using standard methods for histological and immunohistological studies.

RNAi constructs

We performed RNAi experiments using 2 different oligonucleotides targeting the coding sequence or the 3 UTR of rat :

′ Tubb2b
CDS-sh ( 318-341) and 3 UTR-sh ( 1553-1576). A BLAST search against databases confirmed the specificity of each target. Annealed

# ′ #
oligonucleotides were cloned into a mU6-pro vector . For the rescue experiment, we subcloned the coding sequence of [START_REF] Yu | RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells[END_REF] Rattus cDNA (IMAGE 5599369) without the UTRs, into the pCAGIG vector (Addgene plasmid 11159, Cambridge, norvegicus Tubb2b [START_REF] Matsuda | Electroporation and RNA interference in the rodent retina in vivo and in vitro[END_REF] MA), such that a CMV immediate early enhancer/chicken -actin promoter (CAG) drives transcription of the bicistronic Apparatus, Holliston, MA). The voltage was discharged in 5 electric pulses at 950 ms intervals via 5 mm electrodes placed on the head of the embryo across the uterine wall. We performed electroporation in embryonic rats at E15.5. This moment corresponds to an in utero active period of both radial and tangential migration of newborn neurons in the cortex.

Protein modeling

A model of human -tubulin was built by homology modeling using available structures ( Figure 3a http://www.pymol.org

).

translation In Vitro

Transcription/translation reactions were performed at 30 C for 60 min in 25 l of rabbit reticulocyte lysate (TnT T7 Quick Coupled °µ Transcription/Translation System, Promega, Madison, WI) containing 35S-methionine (specific activity 1,000 Ci/ mol; 10 Ci/l) using µ µ

TUBB2B wild-type and mutant cloned into the pET-23b( ) vector (Novagen, Madison, WI) as templates. For the generation of labeled + heterodimers, transcription/translation reactions were chased for a further 2 h at 30 C by the addition of native bovine brain tubulin at 0.2 °mg/ml.

Labeled reaction products were detected by autoradiography after resolution on either SDS-PAGE or on native polyacrylamide gels as described , .

[14 16 ]

Folding Reactions In Vitro

folding assays were performed either directly in the rabbit reticulocyte translation cocktail, or in folding buffer containing In vitro CCT (cytosolic chaperonine), ATP, GTP and tubulin chaperones (TBCB, TBCC, TBCD, TBCE) as described previously

. Target [START_REF] Tian | Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors[END_REF] proteins (i.e. wild type or mutant forms of TUBB2B -tubulin) were expressed as 35S-labeled proteins in BL21 DE3 pLysS cells

β [22 ]
(Invitrogen, Carlsbad, CA) and the inclusion bodies purified and unfolded in denaturant following previously described procedures .

[23 ]

Reaction products were further analyzed by electrophoresis on native polyacrylamide gels , . In some experiments, the yield of [14 16 ] various reaction products was determined using a phosphorimager (Personal Molecular Imager, Bio-Rad Laboratories, Hercules, CA).

Microtubule Co-polymerization Experiments

Products of translation reactions were mixed with depolymerized bovine brain microtubules and taken through successive in vitro cycles of polymerization and depolymerization as described . At the end of each cycle, aliquots containing equal amounts of [START_REF] Tian | A Pachygyria-causing alpha -Tubulin Mutation Results in Inefficient Cycling with CCT and a Deficient Interaction with TBCB { }[END_REF] depolymerized material were removed and analyzed by SDSPAGE and autoradiography.

Cell Culture, Transfections and Immunofluorescence

Constructs were transfected into COS-7 cells grown on glass coverslips in Dulbecco s Modified Eagle s Medium containing 10 (v/v) areas of PMG that appear more severe in frontal and parietal lobes (b,e) and involve the perisylvian region (e,h). The PMG appears either mildly (b,h) or severely asymmetric with left-sided predominance (e) (Hatched lines highlighted by arrowheads show some of the PMG areas). Basal ganglia appear dysmorphic with a fusion of caudate and putamen, and apparent absence of the anterior arm of the internal capsule (b,e,h). Midline sagittal section shows corpus callosum agenesis (c), hypogenesis and abnormal thickness (f), or dysmorphy with a flat shape (i), associated with mild to severe cerebellar vermis hypoplasia or atrophy (c,f,i) and with brainstem hypoplasia (double asterisks).

Axial section at the level of the cerebellum and temporal lobes show severe vermian dysplasia (d,j) or atrophy (g) (black asterisks). (k n,q,r) pathway involves a series of molecular chaperones whose function is to facilitate the assembly of the / tubulin heterodimer . Newly α β [START_REF] Cowan | Type II chaperonins, prefoldin, and the tubulinspecific chaperones[END_REF] translated -tubulin ( ) and -tubulin ( ) polypeptides are first captured and stabilized by prefoldin (PFD) that acts as a shuttling protein to α α β β deliver its bound target protein to the cytosolic chaperonin (CCT) . CCT generates productive quasi-native folding intermediates which [START_REF] Vainberg | Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin[END_REF] interact with a set of downstream Tubulin-specific Chaperones (TBCs) . TBCB and TBCE capture CCT-generated -tubulin intermediates the original level, but declined to an undetectable level in the case of p.F265L. Similar data were obtained in the case of the p.S172P mutation ( ). The level of radioactivity present in complexes at the 1x concentration is taken as 100. Bars represent the average of Supplementary Fig. 8 two experiments. Arrows in (a c) denote the migration positions of the CCT/ -tubulin binary complex, the TBCD/ -tubulin co-complex, the

  All mutations, c.514T C (p.S172P), c.629T C (p.I210T), c.683T C (p.L228P), c.793T C > > > >

β-

  Tubb2b IRES-GFP mRNA. The rescue experiment allows the expression of a 3 UTR targeting shRNA in combination with the overexpression ′ construct. It implies that the overexpressed RNA is resistant to the ShRNA as the target (3 UTR) is absent. Tubb2b′ electroporationIn uteroWistar rats (Janvier, Le Genest Saint Isle, France) were mated, maintained and used in our animal facilities in agreement with European Union and French legislation. Timed pregnant rats embryonic day 15 (E15) E16; E0 was defined as the day of confirmation of [ sperm-positive vaginal plug were anesthetized with ketamine/xylazine (respectively 0.1 and 0.01 mg per g body weight). The uterine ] horns were exposed, and a lateral ventricle of each embryo injected via pulled glass capillaries and a microinjector (Picospritzer II; General Valve Corp., Fairfield, NJ) with Fast Green (2 mg/ml; Sigma, St. Louis, MO) combined with the following DNA constructs: 0.5 μ g/ l pCAGGS-red fluorescent protein (mRFP) either alone or with 1.0 g/ l of shRNA construct targeting the mRNA. Plasmids μ μ μ Tubb2b were further electroporated by discharging a 4000 F capacitor charged to 50 V with a BTX ECM 830 electroporator (BTX Harvard μ

  using the FuGENE6 transfection reagent (Roche Applied Science, Indianapolis, IN). Twenty-four hours post-transfection cells were fixed with 20 C methanol. Immunostaining was performed with a polyclonal anti-FLAG antibody (to visualize protein -°expressed from the construct; Sigma-Aldrich Inc., St. Louis, MO) and a monoclonal anti--tubulin antibody (to visualize the overall α microtubule network; DM1A, Sigma-Aldrich Inc., St. Louis, MO).

Figure 1

 1 Figure 1 Magnetic Resonance Imaging and Histopathology Analysis of Patients with mutations TUBB2B Linear representation of the -tubulin protein showing the position of heterozygous PMG-associated mutations. Representative brain (a) β

-

  Nissl-stained sections of the 27GW fetus (p.S172P mutation) brain display asymmetrical bilateral polymicrogyria (black arrows in k,l) with callosal agenesis. Left and right hemispheres present respectively typical unlayered polymicrogyric cortex (l,n) and focal polymicrogyria with a completely disorganized cortex and radial neuronal heterotopias (black arrows in k,m). Several nodular heterotopic neuron clusters were also observed in both hemispheres. Sections of cortical regions of left hemisphere probed for vimentin (o,p) or stained by Nissl (q,r) respectively show either abnormalities of radial glial fiber organization (p) or a disorganized cortex (r) with neuronal overmigration through the pial basement membrane into the leptomeningeal space (black arrows in r). Sections (o,q) correspond to a 27GW control fetus. M: meninges. Scale bars: (k n) 500 m, (o r) 100 m.

Figure 2 Figure 3

 23 Figure 2Knock-Down of rat Expression by RNAi Alters Neuronal Migration in the Isocortex In Utero Tubb2b Nissl staining on coronal sections of E20 brains reveals the overall organization of the cortex (a) 5 days after electroporation of a RFP (a-i) coding reporter construct either alone (b) or in combination with 3 UTR-sh (c, overlay with Nissl in d), CDS-sh (e), or their corresponding ′ scrambled controls (f,g). A rescue experiment was performed using CAG--IRES-GFP transfected either alone (h, GFP), or combined Tubb2b with 3 UTR-sh (i, GFP). (j,k) Fluorescence intensities reflecting cell positions were converted into gray values and measured across cortices ′ from the VZ to the MZ (j, 3 UTR-sh). k, Bars represent the mean SEM of fluorescence intensities in 10 strata dividing the thickness of ′ ± cortices of independent brains (RFP n=5, 3 UTR-sh n=6, CDS-sh n=6, Scrambled 3 UTR-sh n=4, Scrambled CDS-sh n=5, Rescue n=6). ′ ′ Knockdown of using both hairpins between E15 and E20 disrupts neuronal migration (c,d,e). RFP positive cells are significantly Tubb2b stalled within the deep layers of the cortex (c,k, strata 4,5: F(3,42) 20.4, p 0.0001; p 0.001( ) for 3 UTR-sh compared by Tukey-Kramer > < < *** ′ test to RFP, scrambled 3 UTR-sh and Rescue respectively) that correspond to the SV/IZ (d, higher magnification) whereas neurons have ′ already reached the CP in control conditions (b,f,g). Tubb2b overexpression preserves neuronal migration (h) showing that migration disruption is a specific consequence of RNAi as it rescued the defect (i,k strata 8-10: F(3,42) 23.7, p 0.0001, p 0.001( ) for 3 Tubb2b > < < *** ′ UTR-sh compared by Tukey-Kramer test to RFP and Rescue respectively). Hatched lines in (b,c,e-i) correspond to the outer/ventricular limits of the cortex. Scale bars : 200 m (a-i).µ

  encapsulating GTP-binding pocket (the N-site) is already formed28 producing TBCB/ -tubulin (B/ ) and TBCE/ -tubulin (E/ ) and TBCD capture and stabilize CCT-generated -tubulin intermediates forming TBCA/ -tubulin (A/ ) and TBCD/ β β β β -tubulin (D/ ) cocomplexes. TBCD/ -tubulin (D/ ) and TBCE/ -tubulin (E/ ) converge to form a supercomplex with TBCC (C-D/ E/ ).

  Interaction with TBCC (C) results in the triggering of GTP hydrolysis by -tubulin16. This reaction acts as a switch to signal the release of β newly formed GDP-bound / heterodimers, which are then competent for incorporation into microtubules.Analysis by SDS-PAGE (c) α β (c-e) or non-denaturing gels (d,e) of transcription/translation products performed with wild-type (WT) and mutant TUBB2B and further in vitro chased with bovine brain tubulin so as to generate / tubulin heterodimers (e). The different migration pattern for p.L228P in (c) can be α β explained by the substitution of a proline in place of leucine, disrupting the helix, and presumably resulting in a change in the binding of SDS and hence a slight change in migration rate on the SDS gel. Note that p.F265L and p.S172P mutants yielded either only a trace or no discernable amount of / heterodimer. The remaining mutants all generated products present in the WT control, but in varying yield (d,e).

Figure 4

 4 Figure 4Reactions Reveal a Lowered Affinity of p.S172P for TBCD In Vitro TUBB2B Analysis on non-denaturing gels of the products of reconstituted folding reactions containing ATP, GTP and various combinations of the (a) purified components that are essential to the heterodimer assembly reaction.Analysis on non-denaturing gels of the products of (b c) -

  Research Collaboratory for Structural

	β
	Bioinformatics PDB code 1TUB) from Nogales et al.	, [11 12 ]	. The image in	was rendered using PyMOL (

[START_REF] Nogales | High-resolution model of the microtubule[END_REF]

[START_REF] Nogales | High-resolution model of the microtubule[END_REF]

[START_REF] Nogales | High-resolution model of the microtubule[END_REF]

[START_REF] Nogales | High-resolution model of the microtubule[END_REF]

[START_REF] Nogales | High-resolution model of the microtubule[END_REF]

[START_REF] Nogales | High-resolution model of the microtubule[END_REF]

Ackowledgements:

We thank Dr Fiona Francis for her helpful comments and critical readings of the manuscript, the patients and their parents who contributed in this study, as well as all the colleagues who provided clinical and imaging information. We thank Pr. Renzo Guerrini for providing us helpful advice. We are grateful to Dr Eric Leguern to have allowed KP to develop this project and all the members of Cochin Institute genomic platform, Cochin Hospital Cell Bank, and Isabelle Souville for their technical assistance. This work was supported by funding from AP-HP, INSERM, FRM (funding within the frame of the Programme EQUIPEs FRM 2007) and ANR (ANR Neuro 2005, project A05183KS and ANR-06-NEURO-008-01 contract number RPV06055ASA). XHJ is supported by a PhD. fellowship of the Ministère de l'Enseignement Supérieur et de la Recherche, by a grant for mobility from Université Paris Descartes and an EMBO short-term fellowship (ASTF 66.00-2008) for his work in NYU medical center. KP is a postdoctoral researcher supported by FRM (Fondation pour la Recherche Medicale).

Footnotes:

J.C. coordinated and instigated the study with D.A.K. and J.F.. X.H.J. performed biochemical, cellular and