David Tepfer

Andreja Zalar

Sydney Leach

Survival of Plant Seeds, Their UV Screens, and nptII DNA for 18 Months Outside the International Space Station

Keywords:

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In the present study, plant seeds germinated after 1.5 years of exposure to solar UV, solar and galactic cosmic radiation, temperature fluctuations, and space vacuum outside the International Space Station. Of the 2100 exposed wild-type Arabidopsis thaliana and Nicotiana tabacum (tobacco) seeds, 23% produced viable plants after return to Earth. Survival was lower in the Arabidopsis Wassilewskija ecotype and in mutants (tt4-8 and fah1-2) lacking UV screens. The highest survival occurred in tobacco (44%). Germination was delayed in seeds shielded from solar light, yet full survival was attained, which indicates that longer space travel would be possible for seeds embedded in an opaque matrix. We conclude that a naked, seed-like entity could have survived exposure to solar UV radiation during a hypothetical transfer from Mars to Earth.

Chemical samples of seed flavonoid UV screens were degraded by UV, but their overall capacity to absorb UV was retained. Naked DNA encoding the nptII gene (kanamycin resistance) was also degraded by UV. A fragment, however, was detected by the polymerase chain reaction, and the gene survived in space when protected from UV. Even if seeds do not survive, components (e.g., their DNA) might survive transfer over cosmic distances.

Introduction

T he question of the origin of life has arisen in diverse human cultures. We know now that sequence-based phylogenetic trees are rooted in microbial ancestors, that microbes appeared within the first billion years of Earth's existence [START_REF] Westall | Life on an anaerobic planet[END_REF], and that all organisms, so far examined, use essentially the same genetic code, suggesting a unique origin for life on Earth. These facts are compatible with, but do not prove, an extraterrestrial origin for the life we know.

Habitable zone planets and the basic molecules of life, including water, are found within and beyond our solar system [START_REF] Barman | Identification of absorption features in an extrasolar planet atmosphere[END_REF][START_REF] Eisner | Water vapour and hydrogen in the terrestrialplanet-forming region of a protoplanetary disk[END_REF]. Could life be dispersed among these potential exohabitats? The ability of life-forms to withstand space travel can be used as a measure of the plausibility of this hypothesis. In the SEEDS portion of the EXPOSE-E project, we thus exposed Arabidopsis and to-bacco seeds to space conditions for 18 months outside the International Space Station (ISS), orbiting at 440 km altitude [START_REF] Rabbow | EXPOSE, an astrobiological exposure facility on the International Space Station-from proposal to flight[END_REF] (Fig. 1). Five wild-type and mutant seed genotypes were tested, with each genotype repeated at three dispersed positions (Fig. 2). Behind this fully exposed layer (S1), identical samples (S2) were protected from UV light but exposed to space vacuum, temperatures, and galactic cosmic radiation. In a ground simulation, another set of samples (G1) in an identical sample carrier was subjected to simulated space vacuum, temperature, and UV 200-400nm light, in accordance with data from sensors on EXPOSE, with a dark layer (G2) that lacked UV exposure. Lab controls (L0) were stored at 4°C.

Arabidopsis thaliana was chosen for its small seeds (permitting large sample sizes) and the availability of mutants and information about the composition of the seed coat. Two wild-type Arabidopsis ecotypes, Wassilewskija (Ws-2) and Columbia (Col-0), were exposed, with a mutant lacking a UV screen for each ecotype. Flavonoids and sinapate esters protect plants from UV light [START_REF] Li | Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation[END_REF][START_REF] Stapleton | Flavonoids can protect maize DNA from the induction of ultraviolet radiation damage[END_REF][START_REF] Landry | Arabidopsis mutants lacking phenolic sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage[END_REF][START_REF] Sheahan | Sinapate esters provide greater UV-B attenuation than flavonoids in Arabidopsis thaliana (Brassicaceae)[END_REF], and flavonoids are also antioxidants [START_REF] Pietta | Flavonoids as antioxidants[END_REF][START_REF] Urquiaga | Plant polyphenol antioxidants and oxidative stress[END_REF][START_REF] Williams | Flavonoids: antioxidants or signalling molecules?[END_REF]. The tt4-8 mutant [START_REF] Debeaujon | Proanthocyanidinaccumulating cells in Arabidopsis testa: regulation of differentiation and role in seed development[END_REF] lacked flavonoids due to a T-DNA insertion into the chalcone synthase gene, and the fah1-2 mutant (Columbia background) lacked sinapate esters due to a dysfunctional ferulate-5-hydroxylase [START_REF] Chapple | An Arabidopsis mutant defective in the general phenylpropanoid pathway[END_REF][START_REF] Sheahan | Sinapate esters provide greater UV-B attenuation than flavonoids in Arabidopsis thaliana (Brassicaceae)[END_REF]. Tobacco (Nicotiana tabacum) was chosen for its developed endosperm and redundant genome, in contrast to the vestigial endosperm and the compact genome of Arabidopsis. In addition to the Havana tobacco wild type, a derivative (PT) was tested, carrying a marker in the chloroplast DNA that did not effect phenotype [START_REF] Carrer | Kanamycin resistance as a selectable marker for plastid transformation in tobacco[END_REF].

Flavonoids and DNA were exposed as dry films on the inner surfaces of MgF 2 windows. Quercitrin, the dominant flavonoid in Arabidopsis seeds, has a VUV-UV absorption spectrum that closely matches that of DNA, with additional absorbance in the UVA, while the absorption spectrum of catechin mimics that of protein [START_REF] Zalar | Directed exospermia: II. VUV-UV spectroscopy of specialized UV screens, including plant flavonoids, suggests using metabolic engineering to improve survival in space[END_REF][START_REF] Zalar | Resistance of Arabidopsis thaliana seeds exposed to monochromatic and simulated solar polychromatic UV radiation: preparation for the EXPOSE space missions to the International Space Station (ISS)[END_REF]. DNA from the nptII gene, which encodes kanamycin resistance, was exposed with the expectation that it would be completely degraded by solar UV light.

Materials and Methods

Plant materials and seed germination

With the exception of the fah1-2 mutant (Nottingham Arabidopsis Stock Centre), Arabidopsis seed stocks were from the Versailles Arabidopsis Resource Centre. Tobacco, including Havana PT (pTNH32-70-2), carrying a nptII marker in the chloroplast DNA [START_REF] Carrer | Kanamycin resistance as a selectable marker for plastid transformation in tobacco[END_REF], was a gift from P. Maliga. The PT line was included to provide in planta, multicopy nptII for DNA degradation studies to be described elsewhere. Seed samples were produced in the greenhouse simultaneously for all genotypes of the same species and spread in monolayers behind UV-transparent, 7 • 7 • 1 mm MgF 2 windows [START_REF] Zalar | Resistance of Arabidopsis thaliana seeds exposed to monochromatic and simulated solar polychromatic UV radiation: preparation for the EXPOSE space missions to the International Space Station (ISS)[END_REF]. Seeds were held in place by a steel plate and spring. Mean sample sizes per window were 16.3 -1.74 mg (standard deviation, s.d.) for Arabidopsis and 20.9 -1.38 mg (s.d.) for tobacco. After return to Earth, the sample carrier was stored desiccated at 4°C. Seeds were removed from behind the windows in a laminar flow hood with an insect pin that had been coated with adhesive from double stick tape. The pin was dipped in sterile water, then touched to a seed, which adhered to the film of water but not to the adhesive. Seeds were placed on agar medium, containing 0.8% agar (w/v) in deionized water in three Petri dishes (Fig. 2C), according to their position during exposure so that any exposure gradient would be detected in the sequence of seed germination (dish 1: seeds 1, 4, 7, etc.; dish 2: seeds 2, 5, 8, etc.). For Arabidopsis, 150 seeds from each window were sampled in three batches of 50 seeds in each round of experiments (designated tiers I and II). For the larger tobacco seeds, 50 seeds were removed from each tier in the same fashion and placed sequentially onto a single Petri dish. Each experiment included lab controls (L0) that had been stored at 4°C. Petri dishes were maintained at 8°C in the dark for 78 h to attain homogenous imbibition before the start of germination. Germination took place at 22°C, under 16 h of 4 lE m -2 s -1 light from white compact fluorescent lamps (Philips Softone SW) and red LED lamps (Philips E27G50R). It was monitored hourly during periods of rapid change. Root emergence was scored under 14 • magnification and recorded on the back of the Petri dish with a colored symbol for each time point (Fig. 2C). Samples were identified by numbers. Determination of germination was thus blind, except for the flavonoid-lacking tt4-8 seeds, which were obvious due to their yellow color. Seedlings were removed from Petri dishes and transferred to the greenhouse in squares of agar to avoid damage to the young roots.

Exposed chemicals

Chemical samples included two flavonoid UV screens found in the Arabidopsis seed coat and DNA from the nptII gene, which encodes kanamycin resistance. The dominant Arabidopsis seed flavonoid [START_REF] Routaboul | Flavonoid diversity and biosynthesis in seed of Arabidopsis thaliana[END_REF] is quercitrin (quercetin-3-O-rhamnoside, Sigma-Aldrich). Catechin (+ 3,3¢,4¢,5,7,Extrasynthese,Genay,France) is also an important flavonoid in Arabidopsis. From a 1.5 mg mL -1 solution in 50% (v/v) methanol (HPLC grade), 10 lL of each flavonoid was air-dried on the inside (center) of each of two MgF 2 windows, which corresponded roughly to the tier I + II surface area. Postflight UV absorption spectroscopy (Shimadzu HPS-2000) was performed on these dry films adhering to the inside of the sample windows (in their original positions in the sample carrier).

The DNA sample of the nptII gene was synthesized from a bacterial plasmid template with the polymerase chain FIG. 2. Experimental layout and sampling. (A, Top) The SEEDS experiment on EXPOSE with sample identification numbers (same orientation as in Fig. 1). Large seeds, tobacco; small seeds, Arabidopsis; yellow seeds, Arabidopsis flavonoid minus mutant (tt4-8). Dark-colored samples include chemical sunscreens and DNA, which is in the center of each group of nine samples. (A, Bottom) side view of the sample carrier, inverted with the exposed layer (S1) downward, before compression of the springs. (B) Enlarged view of a typical seed sample (number 313, Arabidopsis Columbia wild type), seen from outside the MgF 2 window. Seeds were held in a monolayer from behind by a steel plate and spring. Yellow dots, tier I; blue dots, tier II. (C) Seeds from each tier (150 for Arabidopsis, 50 for tobacco) were distributed sequentially into three Petri dishes, starting in the center of the window. Petri dishes are shown at the 10-day end point of the germination test, with a symbol marking the time of each germination event. Color images available online at www.liebertonline.com/ast reaction (PCR) and the following primers: 5¢-GAACAA GATGGATTGCACGC-3¢ and 5¢-AGAAGGCGATAGAAGG CGATGC-3¢, which produced a 773 base pair (bp) fragment (nucleotide 7 to 780) from the 795 bp coding region of nptII. The PCR product was purified with the QIAquick PCR purification kit (Qiagen, France). PCR conditions included an initial denaturation step at 95°C for 5 min, followed by 35 amplification cycles consisting of 1 min at 95°C (denaturation), 1 min at 55°C (annealing), and 2 min at 72°C (extension), for 35 cycles, followed by a final extension at 72°C for 5 min. Each of four MgF 2 windows received 10 lL of a 50 lg mL -1 solution of this PCR product in ultrapure H 2 O. After exposure, DNA was resuspended in 2 • 20 lL of TE buffer (10 mm Tris, 2 mm EDTA). DNA survival was qualitatively determined by PCR and the above primers for the 773 bp reaction product (nucleotides 7 to 780), as described above. The presence of a 110 bp internal fragment (nucleotides 79 to 189) was assayed with the above reaction conditions, with primers 5¢-AGACAATCGGCTGCTCTGAT-3¢ and 5¢-CTCG TCCTGCAGTTCATTCA-3¢. PCR products were analyzed by gel electrophoresis in 1.2% (w/v) agarose, followed by ethidium bromide staining.

Further search for biologically active DNA made use of homologous recombination in Acinetobacter baylyi (strain BD413), between exposed nptII DNA and a defective copy of nptII, carried by plasmid pMR7. The defect is a 10 bp deletion starting at coding sequence position 5999 (de [START_REF] De Vries | Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation[END_REF][START_REF] Tepfer | Homologydependent DNA transfer from plants to a soil bacterium under laboratory conditions: implications in evolution and horizontal gene transfer[END_REF]. There were 520 bp between the start of the surviving fragment and the beginning of the deletion. Thus, the 110 bp nptII fragment, detected by PCR after exposure to complete space conditions (see Results), did not include the 10 bp deletion.

Temperature and pressure conditions

The temperature in the sample carrier in space fluctuated 16 times during each of the 558 days of exposure, in accordance with illumination changes during the 91 min orbital period. High-temperature periods (30-40°C for ca. 40 days) alternated with low-temperature periods ( -5°C to -10°C for ca. 20 days) due to changes in the orbital plane of the ISS. The lower limit was determined by heaters that were activated at -12°C. Exceptions were recorded, which were the result of power loss during a low-temperature period and repositioning of the ISS for solar panel installation. The minimum recorded temperature was -25°C and the maximum + 61°C. Temperature fluctuations produced approximately 200 transitions across 0°C, with uncertainty due to loss of 25% of the data. Temperatures were uniform over the two levels of samples. Temperatures in the ground simulation mimicked temperatures in space, with temperatures kept below 30°C during the UV exposure (see below). Pressure in space varied, depending on the orientation of the ISS, between 10 -7 and 10 -4 Pa, according to estimates made with data from the pressure gauge on the MEDET facility, adjacent to EXPOSE on the ISS.

Light conditions

The patterns of temperature change reflect changes in exposure to sunlight, which was determined by shadow mapping (RedShift, Brussels), solar light measurements from the SORCE satellite, and detailed positional information for the ISS (NASA). The UVA, B, C dose for the entire mission was first calculated by accounting only for shadows and reflections from dynamic structures, such as solar arrays and the position of the ISS, but not accounting for static structures, such as open shutters. A total dose of 1.23 • 10 6 kJ m -2 (UVA 315-400nm = 9.58 • 10 5 kJ m -2 ; UVB 280-315nm = 1.95 • 10 5 kJ m -2 ; UVC 100-280nm = 7.76 • 10 4 kJ m -2 ) was thus calculated. This UV dose was then corrected for shadows and reflections from static objects, including the Columbus module, shutters, window frames, and so on, and exposure was simulated at 24 positions within each window. In a typical sample, exposure falloff within tier I was < 10%, and it was < 20% within tier II. The total UV dose is thus best summarized by using fully corrected values for the center of each exposed window: UVA 315-400nm = 5.78 • 10 5 kJ m -2 ; UVB 280-315nm = 1.17 • 10 5 kJ m -2 ; UVC 100-280nm = 4.62 • 10 4 kJ m -2 , for a total UV 100-400nm exposure of 7.4 • 10 5 kJ m -2 . This exposure estimation does not take into account transmission loss through the MgF 2 windows (see below).

UV 200-390 spectroscopy (not shown) through MgF 2 windows that had not been in contact with seeds showed that they were initially transparent to solar UV light down to 110 nm, with no reduction in transmission (measured at 200-300 nm) after exposure. However, the seeds of both species left a residue on the inside of the windows that caused a transmission loss of about 12% at 390 nm and 46% at 200 nm (in a typical window), indicating that the total exposure to UV was less than the value given above. The time course of transmission loss is not known. Thus, transmission decreased during exposure, but not due to inherent changes in the windows.

Seeds in the ground simulation (G1) received 5.8 • 10 5 kJ m -2 of UV 200-400nm light from a solar lamp (SOL 2000, Dr. Ho ¨nle UV technology, Gra ¨felfing, Germany). UV treatment was performed over a period of 1 month, following the temperature plus vacuum simulation.

Radiation conditions

Particle radiation was measured by passive detectors built into the EXPOSE sample carriers 1 and 2, which were adjacent to the SEEDS experiment in carrier 3. The dose was 53.1 mGy galactic cosmic rays (583 days of exposure), 237.7 mGy of South Atlantic Anomaly protons (558 days of exposure), and 4.8 mGy outer radiation belt electrons (558 days of exposure). The total dose behind the outer (S1) MgF 2 windows was 295.6 mGy. The dark (S2) and exposed (S1) layers received the same dose of galactic cosmic rays, but lower-energy particles (electrons and protons) were received primarily by the exposed layer. The dark samples on the starboard side (away from the Columbus module) received approximately 25% more lower-energy particles than samples on the port side, closer to Columbus, probably due to a solar wind shadow, which is similar to the UV shadow caused by Columbus and nearby structures. Ionizing radiation doses are low on the ISS due to protection from Earth's magnetic field.

Statistical analysis

P values were calculated by using the Wilcoxon-Mann-Whitney test in the graphing and statistics program, Kalei-daGraph. P > 0.05 was considered significant.

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Spaceflights and seed storage

The SEEDS experiment was part of EXPOSE-E, which was launched by NASA on 7 February 2008 (flight STS-122), with a return on 12 September 2009 (flight STS-128 Cal). Sample de-integration took place on 3 December 2009. Pre-and postflight temperatures were ambient; preflight seeds were stored in EXPOSE under nitrogen, but postflight they were in ambient atmosphere. After de-integration, seeds were stored in the sample carrier (Fig. 2), desiccated at 4°C.

Results

Seed germination

Seeds were returned to Earth after 558 days of exposure on the EuTEF platform (Fig. 1). The deleterious effects of space travel were quantified by studying their germination. Exposure artifacts were assessed, including edge effects (shading from the edges of the sample windows), position effects (shading from nearby objects), and seed overlap effects (shading from adjacent seeds). Position effects were evaluated for each seed type by comparing similar samples at three dispersed positions on the exposed surface (Fig. 2A). To estimate edge effects, seeds were removed starting at the center of the seed monolayer and working outward in a spiral (Fig. 2B). Germination was scored microscopically as root emergence, with observations made hourly during periods of rapid change. It was expressed as the accumulation of observations of root emergence over time and presented as the mean of the three, 50-seed samples (Figs. 3 and4). Survival was defined as germination that produced morphologically intact, growing plants, scored at the last time point in the germination curve, 10 days after the beginning of imbibition.

An important concern was to assess artifacts due to edge and position effects. Within each tier, no patterns of germination could be attributed to the initial seed position behind the MgF 2 window; that is, for a given Petri dish, seeds closer to the center of the MgF 2 window did not germinate later than seeds away from the center (Fig. 2C). However, in the Arabidopsis Ws-2 wild type and the tt4-8 mutant, germination kinetics and overall survival were improved in tier II, relative to tier I (Fig. 3A,3D). Germination differences between tiers I and II were generally smaller for the other genotypes (Fig. 3), which indicates that edge effects were largely avoided by restricting the sampling to tiers I and II. Data from tiers I and II were thus pooled (Fig. 4), in keeping with the data in Fig. 3 and the prediction from shadow mapping that light falloff was < 20% in tier II.

In some experiments, germination and survival differed among the three 150-seed samples of each seed type, dispersed on the surface of the SEEDS carrier (e.g., Fig. 3C,3D,3F). Since these position effects were not observed in the S2 dark layer (Fig. 5), they were probably due to variable UV microenvironments on the exposed surface, caused by shadows and reflections from nearby objects. However, position variability was greater in Arabidopsis Ws and in tobacco than in Arabidopsis Columbia, which suggests that other factors (including the magnitude of germination, transmission loss in the MgF 2 windows, and conditions in individual Petri dishes) could have contributed to the apparent position effects. Despite these uncertainties, positional effects did not preclude finding statistically valid differences among the genotypes by using data pooled from the three positions tested (Fig. 4).

Modeling (RedShift, Brussels) of the shadow effects predicted an exposure gradient over the exposed samples from left (low) to right (high) and another from bottom (low) to top (high), as the seed sample carrier is presented in Fig. 2A. Correction (not shown) of the raw germination data, with use of the shadow map-derived value for the center of each sample position, did not alter the conclusions drawn from the uncorrected data. Thus, the data presented in Fig. 3 are not corrected for uneven illumination.

Survival results (maximum seed germination that produced intact plants) were pooled from tiers I and II and from the three positions on the surface of EXPOSE (Fig. 4). Pooled data for tt4-8 and tobacco were only corrected for differences in lab control germination, evident in the uncorrected L0 mean data (Fig. 3D). Correction factors were 1.087 for tobacco and 1.064 for tt4-8. The sample size (batches of 50 seeds) for each genotype was 900 seeds in Arabidopsis and 300 in tobacco. The Havana and PT tobacco survival results were also pooled, since no phenotype is associated with PT, and there was no statistical difference in germination between the two lines (P = 0.559).

The germination kinetics and survival of wild-type seeds varied with genotype (Figs. 3 and4). The highest survival was 44% (n = 1 • 50 seeds) in tobacco (Havana), tier II, position 293 (Fig. 3F). Among the wild types, WS was less resistant than Columbia (Fig. 3A, 3B, 3D, 3E, Fig. 4), and survival in tobacco was similar to that in Columbia (Fig. 3B, 3C, 3E, 3F, Fig. 4). Arabidopsis mutants lacking sunscreens were less resistant to full exposure than the corresponding wild types (Figs. 3 and4). No tt4-8 (flavonoid minus) seeds germinated in tier I (Fig. 3A, Fig. 4). Lack of sinapate ester sunscreens ( fah1-2 mutant) was also associated with reduced germination, but to a lesser extent than in tt4-8 (Figs. 3 and4). Survival thus varied with ecotype and was improved by UV screens.

Radiation effects

Comparison between the dark layers in the ground simulation (G2) and those on the ISS (S2) allowed an estimate of galactic cosmic radiation effects in the dark layer in space, since the ground simulation dark layer received equivalent space vacuum and temperature but was spared the cosmic radiation. (Solar ionizing radiation was largely stopped by the S1 layer.) The onset of germination was delayed by 7 h in Arabidopsis Columbia in the dark layer exposed to galactic cosmic radiation, compared to the lab control and ground simulations (Fig. 5). In tobacco, germination was delayed by 14 h. However, total survival in wild-type Arabidopsis and tobacco (pooled Havana + PT) did not differ in the dark layers on the ISS and on the ground (Fig. 6). Thus, galactic cosmic radiation was a minor, but measurable, liability during the 18-month exposure. Nevertheless, it could cause severe damage during longer journeys through space [START_REF] Kranz | Biological damage induced by ionizing cosmic rays in dry Arabidopsis seeds[END_REF][START_REF] Wei | Analysis of cytogenetic damage in rice seeds induced by energetic heavy ions on-ground and after spaceflight[END_REF]. In contrast to the other genotypes, survival of the tt4-8 mutant (lacking flavonoids) was reduced in the dark layers, both in space and on the ground (Fig. 6), which possibly reflects an additional role for flavonoids in responses to temperature fluctuations ( -25°C to + 61°C).

FIG. 3.

Germination kinetics for samples exposed on the ISS to complete space conditions (S1). Black circles, wild-type lab controls; white circles, flavonoid minus mutant lab controls (L0); black squares, exposed wild types; white squares, exposed flavonoid minus mutants. For Arabidopsis, each curve represents 150 seeds, summarized as the mean of three curves, each derived from 50 seeds (Fig. 1). For exposed tobacco, each curve represents one 50-seed sample; control curves represent the mean of four 50-seed samples. Germination time: [The delay in germination in (A) is an artifact caused by unintended imbibition at 4°C rather than 8°C.]

Temperature and vacuum effects

Survival was similar in the Ws lab control (L0) and in the space dark control (S2), which indicates that exposure to space temperature and vacuum did not diminish survival (Fig. 6). However, survival was reduced in the tt4-8 seeds exposed to space temperature and vacuum WS (S2) 98.1 -2.2; n = 18; tt4 (S2) 84.6 -7.4; n = 18; P = < 0.0001. In contrast, survival in tobacco increased (Fig. 6) by six percentage points in seeds exposed to space vacuum and temperature (G2): lab control (L0), 91.1% -6.33, n = 8 • 50; ground simulation (G2) 97.2% -2.33; n = 12 • 50; P = 0.015. Space temperature and vacuum thus stimulated survival in tobacco, perhaps due to better desiccation in space. We conclude that after 18 months of exposure, radiation and vacuum plus temperature effects could be measured, but they were minor, species dependent, and not necessarily deleterious. Data were corrected for tt4-8 (multiplication factor, 1.064) and tobacco (multiplication factor, 1.087) to normalize for inherently lower germination in the respective lab controls (Fig. 3C,3D,3F). Data from the three repeats of each genotype were pooled, and tobacco data include Havana plus the PT insertion line.

Phenotype of survivors

To assess the morphology and fertility of the survivors at the end of the 10-day germination test, 10 Arabidopsis plants or five tobacco plants (or ungerminated seeds, when germination was low), representative of each sample of 50 seeds, were transferred on blocks of agar to soil in the greenhouse and grown through seed set. Morphological abnormalities were not evident in Arabidopsis and tobacco in vitro, and the majority of the plants of both species were capable of growth and seed set in the greenhouse. However, slow growth and reduced seed set were common in Arabidopsis exposed to space UV (S1), but not in the dark layer (S2). In tobacco, slow growth and reduced seed set occurred in seven out of the 101 plants grown to maturity from exposed seeds (S1), with one plant dying upon transfer from agar to soil. The reduced effects of exposure in tobacco, compared to Arabidopsis, could be due to the more developed endosperm in tobacco, which provides better shielding of the embryo, and perhaps to tobacco's larger, more redundant genome.

Phenotype in the first sexual generation after exposure

Seeds were sown from Arabidopsis Ws (S1) tier I survivors of full space exposure, the most affected by exposure (Fig. 3A). All 44 seeds germinated, and 42 produced fertile plants with a normal seed set. One plant was of small stature, and one plant produced only one seed. No morphological aberrations were observed. Thus, the reductions in growth and fertility seen in the survivors of full exposure to space largely disappeared in the first sexual generation after exposure. A similar result was obtained in tobacco. Survival thus appears to have been an ''all or none'' phenomenon; the survivors' genome was not irreparably damaged, and failure of germination was likely due to general UV damage due to other factors, including a lethal accumulation of free radicals, destruction of membranes, or massive damage to ribosomes [START_REF] Casati | Crosslinking of ribosomal proteins to RNA in maize ribosomes by UV-B and its effects on translation[END_REF].

Effects of solar UV on flavonoid UV screens

Flavonoids contributed to the resistance of Arabidopsis seeds to UV light (Figs. 3 and4), but to be effective, their shielding capacity should not be lost during exposure. Comparison of UV 200-390nm absorption by quercitrin and catechin in the fully exposed S1 layer and the dark S2 layer revealed changes in UV absorption in flavonoids from the S1 layer, including a loss of features, a shift to lower wavelengths, and a 30% increase in the capacity of both flavonoids to absorb energy in the region between 200 and 400 nm (Fig. 7). Protection was not lost in the DNA-absorbing regions (200 and 260 nm), but there was a pronounced loss of UVA absorption in the exposed (S1) quercitrin.

Effects of solar UV on nptII DNA

The polymerase chain reaction (PCR) was used to monitor survival of naked DNA encoding a 773 bp region of nptII, a bacterial kanamycin resistance gene. In DNA from the fully exposed S1 layer, the entire 773 bp nptII fragment was not detected by PCR (not shown), but an internal (110 bp) fragment was amplified (Fig. 8A) with another primer set designed to amplify nucleotides 79-189. Strong amplification was detected in two of the four DNA samples. DNA amplification of this short fragment was therefore verified in a second round of PCR by using 1 lL from the first round. The FIG. 4. Survival according to genotype. Sample repeats were pooled by using each 50-seed sample from Fig. 3 (sample repeats and tiers I, II). To enable comparison, pooled data were corrected for tt4-8 (multiplication factor, 1.064) and tobacco (multiplication factor, 1.087), thus normalizing for inherently lower germination in the respective lab controls (Fig. 3C,3D). Both flavonoid-and sinapate-lacking mutants were less resistant than the corresponding wild types, and the Ws wild type was less resistant than Columbia. Data are presented as meanss. second round produced strong amplification of the 110 bp fragment in all exposed samples but not in the negative control (not shown). The DNA samples were thus partially degraded, but given the presence of weak signals in two of the four samples, we can infer that template was probably limiting, e.g., that few intact copies of the 79-189 region survived exposure to solar UV light. In contrast, DNA from the S2 layer, which was protected from UV, provided a consistently strong amplification template for the 110 bp fragment (Fig. 8A). Amplification of the 773 bp fragment occurred, and the template DNA was physically intact, as shown by agarose gel electrophoresis (Fig. 8B).

The survival of biologically active nptII DNA was also assessed by using homologous recombination between exposed nptII and a resident, defective nptII, carried by pMR7 in Acinetobacter baylyi strain BD412 (de Vries and [START_REF] De Vries | Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation[END_REF]. Dark layer (S2) DNA (15 ng) from sample position 285 was tested for the ability to confer kanamycin resistance, that is, to rescue the mutation in the resident nptII, producing 10 6 colony-forming units on medium containing kanamycin. In contrast, no resistant colonies formed when using a 20-fold excess of DNA from the exposed (S1) layer. This biological test for DNA survival thus confirmed the above PCR results: DNA exposed to UV was largely degraded and biologically inactive, but DNA survived in the dark layer, protected from UV light.

Discussion

Experimental variability and seed survival

Variables that might explain heterogeneous responses in the seed population include seed genetic makeup, size, FIG. 5. Cosmic radiation and seed germination kinetics. Effects of cosmic radiation were assessed by comparing germination kinetics in seeds from the dark layer in space (S2), which received galactic cosmic radiation, and seeds from the dark layer in the ground simulation (G2), which did not. (A) Germination kinetics in Arabidopsis (Columbia). Blue curves: seeds from the ground simulation (G2) dark layer, without UV and radiation but with space vacuum and temperatures; red curves: seeds from the ISS dark layer (S2), without UV but exposed to galactic cosmic radiation; black curves, lab controls (L0). Galactic cosmic radiation exposure was associated with a 7 h delay in germination. Each curve represents accumulated germination events, expressed as the mean of three samples, each involving 50 seeds. (B) Same as (A), but with tobacco seeds (Havana plus PT), showing a 14 h delay in germination. Each curve represents a single sample, comprised of 50 seeds. Color images available online at www.liebertonline.com/ast FIG. 6. Effects of cosmic radiation, space vacuum, and temperature on survival. Black columns, lab controls (L0); gray columns, dark controls in space (S2); light gray columns, ground dark controls (G2). Survival was similar in the dark in space and in the corresponding ground simulation (S2 vs. G2) for Arabidopsis Ws (wild type), tt4-8 (flavonoidlacking) and tobacco (Tob), showing that galactic cosmic radiation did not diminish survival. *P < 0.05, ***P < 0.001. compensate for differences between individual seeds and their orientation. We conclude that it is unlikely that interseed shading artifacts caused the survival observed, although they could contribute to the variability among repeated samples from different positions (different windows). Similar arguments can be made for the importance of individual genetic differences, seed size, and seed orientation. The deposit of UV-absorbing residues on the inside of the MgF 2 windows was variable (results not shown), and it could explain some of the differences among sample repeats. Again, experimental variability did not preclude finding differences among the samples predicted by the biology; mutants lacking UV screens were more sensitive to UV than the corresponding wild types.

Radiation effects

Arabidopsis and tobacco seeds received 253 mGy, mostly in the form of protons. Thirteen days of exposure to space radiation produced measurable damage in Arabidopsis seeds [START_REF] Kranz | Biological damage induced by ionizing cosmic rays in dry Arabidopsis seeds[END_REF]. In soybean seeds, effective mutagenesis (DNA deletions) was accomplished at fast neutron doses ranging between 4 and 32 Gy [START_REF] Bolon | Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean[END_REF]. However, cytological aberrations were observed in root tips from rice seeds exposed to space low-orbit radiation for 18 days [START_REF] Wei | Analysis of cytogenetic damage in rice seeds induced by energetic heavy ions on-ground and after spaceflight[END_REF]. Since our seeds were exposed in the dry state, repair mechanisms were not functioning, and accumulated lesions were repaired only after hydration; however, in the dry state fewer free radicals are expected from the radiolysis of water. We conclude that radiation effects were minor, as expected from the doses received, and that the inhibition of germination in both seed species in the exposed layer (S1) was primarily caused by UV light. However, cosmic radiation most likely caused the species-dependent delay in germination (Fig. 5) observed in the dark layer (S2).

UV screens and DNA

Overall absorbance was maintained after exposure of quercitrin and catechin to solar UV, but it was shifted to shorter wavelengths, presumably due to the degradation of these complex molecules into subunits of variable composition, producing a loss of features in the UV absorption spectra. The retention of overall UV absorbance may explain, in part, the role of flavonoids in the observed survival of Arabidopsis seeds. Retention of absorbance in the 200-400 nm region is compatible with the protection of DNA and RNA, and it could partially explain the dearth of morphological mutants among the survivors.

Three methods were used to assess DNA survival. The first, agarose gel electrophoresis, showed that the nptII gene was lost in the S1 layer, exposed to UV, but that it survived in the S2 layer, protected from UV. The second method, PCR, failed to amplify nptII in samples from the S1 layer, although a 110 bp subregion was detected (Fig. 8A). Thus, some DNA fragments survived exposure to complete space conditions, but intact genes did not. The third method, a bacterial transformation and recombination assay for biologically active nptII DNA, also gave a negative result for the S1 layer. However, in the S2 layer, which was protected from UV, the DNA was biologically active, that is, it was taken up by a bacterium, and it could correct a 10 bp deletion in a resident nptII.

We conclude that DNA fragments included in, or adhering to, small particles (e.g., clays) blown into the upper stratosphere by winds or volcanoes could survive if they were not exposed to solar UV light. They might later return to Earth as micrometeorites and re-enter the biosphere through bacterial genetic transformation and recombination with resident sequences, as in the nptII model used here. Transfer of such protected DNA might also operate over cosmic distances.

Seeds as vectors for life

To put the observed seed survival in perspective, we can ask whether a seedlike entity could survive the UV exposure associated with transfer between planets, for example, via ejection caused by a meteorite impact on Mars and transfer to Earth. Given an estimated exposure of 1089 solar constant hours for a seed tumbling at random on a direct voyage (4752 h) and the survival of Arabidopsis (Columbia) measured at 32 -5.6% s.d. (n = 900 seeds) after 1913 solar constant hours of exposure, we can speculate that some of the seeds launched from Mars would survive exposure to UV, temperature extremes, and vacuum during the (6 months minimum transit time) trip to Earth [START_REF] Gladman | Martian meteorite transfer[END_REF][START_REF] Gladman | Destination: Earth. Martian meteorite delivery[END_REF]. However, they would suffer from other stresses, for example, radiation and pressure plus heating upon ejection from Mars and entry through Earth's atmosphere [START_REF] Jerling | Survival of seeds in hypervelocity impacts[END_REF]. Furthermore, seeds were immobilized on EX-POSE. A tumbling seed might respond differently to UV radiation. The chances that a naked seed could survive interplanetary transfer are thus difficult to evaluate. The survival observed here nevertheless demonstrates that, although unprotected seeds can resist prolonged exposure to space, UV light is particularly deleterious. If seeds were encased in an opaque matrix (e.g., including water ice), they would be protected from UV light and perhaps some of the shock of ejection and entry. It is thus conceivable, but not proved, that a seedlike entity could survive transfer from Mars to Earth. Comets could be carriers of both water and life [START_REF] Delsemme | Cosmic origin of the biosphere[END_REF][START_REF] Hoyle | Comets-a vehicle for panspermia[END_REF].

Seeds too damaged to germinate could still release macromolecular components of life into a sterile foreign environment and perhaps jump-start the formation of life [START_REF] Tepfer | Plant seeds as model vectors for the transfer of life through space[END_REF]. Plant seeds harbor endosymbionts, and they sometimes carry free-living bacteria. Thus, seedlike entities could serve as vectors for embedded microorganisms, even if the plant embryo does not survive [START_REF] Tepfer | Plant seeds as model vectors for the transfer of life through space[END_REF].

Seeds are model extremophiles, whose biological properties might be exploited to build a life-form capable of dissemination in large numbers over long distances through space [START_REF] Tepfer | The origin of life, panspermia and a proposal to seed the Universe[END_REF]. For instance, seed coat UV screens could be augmented [START_REF] Yu | Metabolic engineering to increase isoflavone biosynthesis in soybean seed[END_REF][START_REF] Chaudhuri | Transgenic mimicry of pathogen attack stimulates growth and secondary metabolite accumulation[END_REF], and microorganisms with improved DNA repair could be embedded in seeds, which could be coated with nutritive and protective substances. A 20 kg payload would carry about one billion Arabidopsis seeds. Plant seeds and embedded microorganisms could thus be used by humans to disseminate Earth's life toward exohabitats, in a reversal [START_REF] Tepfer | The origin of life, panspermia and a proposal to seed the Universe[END_REF] of the directed panspermia envisioned by Thomas Gold, cited by Sagan [START_REF] Shklovskii | Intelligent Life in the Universe, Holden-Day[END_REF], and by Crick and Orgel [START_REF] Crick | Directed panspermia[END_REF].

526

TEPFER ET AL.

This article has been cited by:

1. Elke

FIG. 1 .

 1 FIG. 1. EXPOSE and SEEDS on the Columbus module of the ISS (Photo courtesy of NASA). EXPOSE (small red box) is on the EuTEF platform. SEEDS is covered by a protective shutter (closed in this photo, taken prior to the start of exposure). Columbus, the European module, is to the left of EXPOSE. Proximal sources of UV and solar wind shadows include Columbus, the other experiments surrounding EXPOSE on the EuTEF platform, and the protective shutter, which was perpendicular to the surface of SEEDS during exposure but closed during transport. Insert (large red box), position of SEEDS on EXPOSE, with the shutters (Sh) open, in the same orientation as in Fig 3, with seeds on the lower right. White arrow, direction of flight, except during shuttle docking, when it was inverted 180°. The external dimensions of EXPOSE were 440 • 380 • 250 mm. Color images available online at www.liebertonline.com/ast

  hours after transfer of seeds to 22°C. (A-C) tier I experiments, (D-F) tier II (see Fig. 2B for tier positions). Numbers indicate sample positions (Fig. 2A). (A, D) Lab Ws, Arabidopsis wild-type Ws lab control (L0); W, exposed Ws wild type; T, tt4-8 (flavonoid minus) Arabidopsis insertion mutant. (B, E) Lab C, Arabidopsis Columbia wild type; F, fah1-2 (sinapate ester minus) Arabidopsis mutant. (C, F) H, tobacco Havana wild type; P, tobacco line PT (nptII marker in the chloroplast DNA, with no associated phenotype).

  FIG. 4. Survival according to genotype. Sample repeats were pooled by using each 50-seed sample from Fig. 3 (sample repeats and tiers I, II). To enable comparison, pooled data were corrected for tt4-8 (multiplication factor, 1.064) and tobacco (multiplication factor, 1.087), thus normalizing for inherently lower germination in the respective lab controls (Fig. 3C, 3D). Both flavonoid-and sinapate-lacking mutants were less resistant than the corresponding wild types, and the Ws wild type was less resistant than Columbia. Data are presented as meanss.d. *P < 0.05, ***P < 0.001. n = 6 • 50 for Arabidopsis lab controls; n = 8 • 50 for tobacco lab controls; n = 18 • 50 for Arabidopsis exposed seeds; n = 6 • 50 for tobacco-exposed seeds. The total calculated UV 100-400nm exposure was 7.4 • 10 5 kJ m -2 .

  FIG. 7. Effects of solar UV light on two Arabidopsis flavonoids, examined with UV absorption spectroscopy (dark lines, S2 dark layer; gray lines, S1 fully exposed layer) (A) Quercitrin. (B) Catechin. Structural features were lost, but overall absorption capacity was retained.

  Rabbow , Petra Rettberg , Simon Barczyk , Maria Bohmeier , André Parpart , Corinna Panitz , Gerda Horneck , Ralf von Heise-Rotenburg , Tom Hoppenbrouwers , Rainer Willnecker , Pietro Baglioni , René Demets , Jan Dettmann , Guenther Reitz . 2012. EXPOSE-E: An ESA Astrobiology Mission 1.5 Years in Space. Astrobiology 12:5,[374][375][376][377][378][379][380][381][382][383][384][385][386]

Acknowledgments

We are grateful to E. Rabbow and the German Aerospace Center (DLR) for ground simulations and sample integration; to T. Beuselinck and C. Van Bavinchove (RedShift Design and Engineering, Brussels) for shadow maps, light exposure simulations, and the Mars-to-Earth exposure estimate; to T. Hoppenbrouwers for the temperature history; to R. Demets, P. Baglioni, and J. Dettmann (ESA) for EXPOSE project management, logistics, and calculations; to the engineers at Kayser-Threde and RUAG for the design and construction of EXPOSE; to NASA for deployment and retrieval; to M. Crespin and M. Romaniuk for greenhouse help; to P. Maliga, C. Chapple, J. de Vries, and W. Wackernagel for biological materials; and to T. Berger and T. Dachev for radiation dosimetry. Funding was from the Centre National d'E ´tudes Spatiales (CNES) and from the European Space Agency (ESA). D.T. designed the sample wells, performed the germination, flavonoid, and DNA studies, and analyzed the data; A.Z. prepared the chemical samples and seed monolayers; S.L. carried out computational analysis; D.T. and S.L. initiated the study; D.T. and A.Z. finalized its design; D.T. wrote the paper.

Author Disclosure Statement

No competing financial interests exist.

Abbreviations bp, base pair; ISS, International Space Station; PCR, polymerase chain reaction; s.d., standard deviation.


Jacques Cabaret
email: cabaret@tours.inra.fr

We intended to relate the geographic distribution of ruminant gastrointestinal nematodes in relation to steppe climate (and vegetation). Data are either from literature or from newly acquired/ available results. Simple or more sophisticated meteorological indices were used to characterize the climate. Regression analyses were used to correlate climatic factors and presence of endoparasites from steppe areas. The distribution of one (Marshallagia) out of five endoparasite genera was concentrated mostly in steppic areas whereas other species were found also in other areas. In wild hosts the distribution of Marshallagia was much larger from Sptizberg to New World (northern territories in Canada or extreme south of America). In domestic small ruminants the presence of Marshallagia was identified more frequently and constantly in the area of original domestication and its early diffusion (from Northern Africa to Kashmir, Caucasia). The distribution of this parasite was correlated to low rainfalls which were not the case for all other endoparasites. After host switch (reindeer or south America camelids), it has expanded in other climatic areas, either colder or dryer.

KEY WORDS: small ruminant, endoparasite nematode, Marshallagia, climate, steppe, host switch.

Résumé

INTRODUCTION

I t may look surprising that parasites would display macroecological and biogeographical patterns similar to those described for free-living organisms (Gueguan, 2006). In fact one may observe in many macroparasites similar ecological patterns as observed for terrestrial or marine free-living taxa [START_REF] Poulin | Parasite biodiversity[END_REF][START_REF] Fernandez | Not everything is everywhere: the distance decay of similarity in a marine host-parasite system[END_REF]. Among the gastrointestinal parasite nematodes of herbivores, one part of the life is non-parasitic on the grass and they are clearly influenced by climatic environment [START_REF] Walkden-Brown | Ecology of the free-living stages of major trichostrongylid parasites of sheep[END_REF]. The main difference is that their life within a host, who ingested infected herbage, is regulated to some extent by host susceptibility [START_REF] Gaba | The establishment rate of a sheep nematode: revisiting classics using a meta-analysis of 87 experiments[END_REF]. One major difference in domesticated hosts is that they are often treated with anthelmintics, which may alter in the long run the diversity of macroparasites [START_REF] Silvestre | Relationship between helminth species diversity, intensity of infection Original contribution Parasite, 2011, 18, 261-269 and breeding management in dairy goats[END_REF]. A second difference in these domesticated hosts is that they are transported from one site to another by their owner, sometimes on long distances for commercial reasons or availability of food. It could then be expected that ecological traits remains unexplainable due to these confounding effects of human management. A previous study on the importance of climate and host species on occurrence of some macroparasitic nematodes (Ostertagiinae) of ungulates (mostly domestic ones and from 585 references altogether) did show that host species and climate played apparently a similar role (18 % of occurrences explained equally either by range of host species or climates) [START_REF] Suarez | Similarities between species of the Ostertagiinae (Nematoda: Trichostrongyloidea) in relation to host-specificity and climatic environment[END_REF]. Interspecific interactions in the gastrointestinal community are limited [START_REF] Cabaret | Comparative analysis of two methods used to show interspecific interactions in naturally acquired parasite nematode communities from the abomasums of ewes[END_REF] and this may simplify the understanding of climate/host factors. One of the genera of macroparasitic nematodes, Marshallagia sp., was associated with steppic climate and with domestic sheep and goats. However we know that it may be present in muskoxen or reindeer under polar climate (Halvorsen & Bye, 1999, in the Spitzberg) and in wild ungulates in several part of mountainous Europe (Italian Alps, among others: [START_REF] Zaffaroni | Host specificity of abomasal nematodes in free ranging alpine ruminants[END_REF]. The steppe climate comes under Köppen's BS classification of climates [START_REF] Viers | Éléments de climatologie[END_REF]. The B stands for dry climates, and the S for steppe climate. There is a huge difference between summer (up to 30 °C monthly temperature) and winter (sometimes below 0 °C monthly temperature) and the differences between day and night are also great. Some mediterranean climates (portuguese and hellenic) are not highly different from steppe climate but the number of dry-month is smaller. Steppe regions are often found in middle of continents and in the lee of mountains. Steppe vegetation may be found under different climates, and this renders the term steppe equivocal. Vegetation often consists in small xerophytic discontinuous grassland cover (opposed to prairie with continuous grass cover or savannah with tall grass). Steppe vegetation thus can be found in regions with very cold winter and hot summers (Central Asia, Eastern Europe) or under subarid mediterraneans climates (North Africa and several Middle-East countries). Thus steppe, either defined by climate or vegetation is not a very clear concept and we will substitute simple measures such as annual mean temperature or yearly rainfall in areas where Marshallagia is highly represented, since it was associated with steppe vegetation in a previous analysis [START_REF] Suarez | Similarities between species of the Ostertagiinae (Nematoda: Trichostrongyloidea) in relation to host-specificity and climatic environment[END_REF]. Nematodirus species are also highly prevalent in steppe regions but they are also found in many different climatic areas [START_REF] Morgan | Agricultural restructuring and gastrointestinal parasitism in domestic ruminants on the rangelands of Kazakhstan[END_REF]. Genus such as Haemonchus (from temperate down to tropical areas where it predominates) or Teladorsagia (not found under the tropics but well adapted to temperate or cold climates) are found under a large range of climates; Trichostrongylus genera is nearly present under any type of climate. For example, North Africa has a large part of the inland under typical steppe climate and vegetation [START_REF] Slimani | 30 years of protection and monitoring of a steppic rangeland undergoing desertification[END_REF], and as expected, Marshallagia has been very frequently recorded (Morocco: [START_REF] Cabaret | Seasonal changes in the abomasal nematodes of naturally infected ewes in Moulay-Bouazza (Morocco)[END_REF]Algeria: Bentounsi et al., 2007 and[START_REF] Boulkaboul | Parasitisme interne du mouton de race Ouled Djellal en zone semi-aride d'Algérie[END_REF][START_REF] Boulkaboul | Parasitisme interne du mouton de race Ouled Djellal en zone semi-aride d'Algérie[END_REF]; but other trichostrongylid nematodes such as Teladorsagia circumcincta, Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Trichostron-gylus probolurus and Nematodirus filicollis are also frequently recorded [START_REF] Graber | Bibliographie des parasites internes des animaux domestiques et sauvages du Maghreb, du Sahara et de la Mauritanie[END_REF].

We intend to answer to the following questions: 1) is Marshallagia associated to particular climatic characteristics such as average temperature and annual rainfalls or rather to a set geographical locations in domestic sheep and goats? 2) is Marshallagia associated with other nematode species in sheep and goats? 3) could the wild hosts infection with Marshallagia help to decipher the distribution of this species?

MATERIALS AND METHODS

PARASITOLOGICAL METHODS

P arasitological examinations concern faeces or gastrointestinal tract of necropsied hosts. The differentiation of eggs in faeces is limited to Marshallagia, Nematodirus and other gastrointestinal nematodes (MAAF, 1986). The worms collected at necropsy are identified to species [START_REF] Skrjabin | Trichostrongylids of animal and man[END_REF]. The organs examined were abomasa (glandular stomach of ruminants) or abomasa and small intestines.

RECORDED OCCURRENCES OF MARSHALLAGIA MARSHALLI IN LITERATURE

The occurrences were obtained from older records [START_REF] Skrjabin | Trichostrongylids of animal and man[END_REF] or more recent records using Web of Science -All data bases in May 2010. We also used parts of the records from [START_REF] Suarez | Similarities between species of the Ostertagiinae (Nematoda: Trichostrongyloidea) in relation to host-specificity and climatic environment[END_REF], that were based on Helminthological Abstracts (1960[START_REF]Estudio epidemiológico sobre los nematodos gástricos ovinos de la provincia de León, con especial referencia a Ostertagia spp[END_REF], Index Catalogue of Medical and Veterinary Zoology (1966)(1967)(1968)(1969)(1970)(1971)(1972)(1973)(1974)(1975)(1976)(1977)(1978)(1979)(1980)(1981)(1982). It was a critical review of occurrences and the cattle occurrences were discarded since either [START_REF] Skrjabin | Trichostrongylids of animal and man[END_REF] or ourselves in our unpublished investigations could not find Marshallagia in cattle. These occurrences are shown in Fig. 1A,B.

OWN SURVEY IN ALGERIA

The studied farm (el Mader) is located in the North-East of Algeria (Department of Batna). The coldest month is January with 5.6 °C. The hottest month is July with 27.5 °C. Yearly rainfalls are 368 mm and the drought period extends from June to September. The climate is categorized as semi-arid with temperate winter or steppic. 30 weaned lambs were selected among the available lambs and were used as sentinels to monitor the infection. They were monthly examined for faecal egg counts and two to three

META-ANALYSIS OF NEMATOFAUNA IN STEPPE AREAS

The sites were selected on the basis of constant presence of Marshallagia and detailed results that encompasses several seasons. The sites were also representative of a large geographic range with Marshallagia. Nine sites or periods were investigated regarding sheep faecal egg counts (Eastearn and western Algeria, Northwestern areas of China, Center area of Kazakhstan, and four areas of Northwestern Syria) with Marshallagia prevalence ranging from 10 (Kazakhstan) to 85 % (our survey in Algeria) (Table I). Necropsies of hosts were performed to identify nematodes found in abomasa in nine sites or periods (Morocco, Mongolia, Kashmir, Syria, Spain) (Table II). The percentage of Marshallagia in the abomasum nematode community ranged from 3 (year three in Morocco) to 81 (Syria 4). Necropsies of hosts on all the digestive-tract were conducted in few sites (Algeria, Kazakhstan, Turkey-Van and Syria) and prevalences are shown (Table III).

CLIMATOLOGICAL PARAMETERS AND ANALYSIS OF DATA

The rainfalls (R) are below 40 cm per year in steppe climate. The rainfalls in steppe climates are related to yearly average temperatures (t in °C) in the following ways: R in cm < 2 t when dry season occurs in summer, R < (t + 14) when dry season is in winter and R < 2 (t + 7) when no season effect is observed. We used the difference between R -2 t, R -(t + 14) or R -(2 t + 14) as an index of departure from steppe climate either in dryness (negative values) or excess of humidity (positive values) and we call it DS (departure from steppe). The lowest value was in, Syria (-12) and the highest in León 4 (53). The De Martonne's aridity index I is calculated as follows: I = R in mm / (t + 10) and is between 5 to 10 for dry steppe. It ranged from 7.2 (Syria) to 35.7 (León 4). All the statistical analyses were performed using SPSS 11.5 (regressions) or MVSP 3.1. (cluster analyses based on UPGMA and Spearman rank correlations) softwares.

RESULTS

GEOGRAPHIC DISTRIBUTION OF MARSHALLAGIA IN WILD AND DOMESTIC UNGULATES

T he distribution of Marshallagia in wild ungu- lates (Fig. 1A) was relatively extended among continents but restricted to few sites. Most of the distribution was associated to mountains except in reindeer from Spitzberg. The wild ungulates disseminated Marshallagia into polar or boreal climates. The infected domestic sheep and goats (Fig. 1B) were also recorded in mountain regions either in the New World (Rocky mountains and Appalachians in North America and the Andes in South America) or in the Old World (Alps, Atlas, Taurus, Caucasus, Himalaya, and Altai). The majority of occurrences were located in Eurasia (45 out of 51 sites) of which half were in the original areas of domestication (Middle-East and Northern India).

NEMATODE FAUNISTIC SIMILARITY BETWEEN SITES AND PERIODS

The faecal egg counts provide a first gross evaluation on the nematode fauna (Table I [START_REF] Cabaret | Seasonal changes in the abomasal nematodes of naturally infected ewes in Moulay-Bouazza (Morocco)[END_REF][START_REF] Sharkhuu | Helminths of goats in Mongolia[END_REF][START_REF] Tariq | Gastro-intestinal nematode infections in goats relative to season, host sex and age from the Kashmir valley, India[END_REF]Giangaspero et al., 1992[START_REF]Estudio epidemiológico sobre los nematodos gástricos ovinos de la provincia de León, con especial referencia a Ostertagia spp[END_REF] Average

Reference

Site

Variance to mean ratio of prevalences Algeria (Batna) Kazakhstan Turkey (Van) Syria 5 Syria 6

Reference

Present study [START_REF] Morgan | Agricultural restructuring and gastrointestinal parasitism in domestic ruminants on the rangelands of Kazakhstan[END_REF][START_REF] Cengiz | Sheep trichostrongylidosis in Van province[END_REF][START_REF] Hörchner | Zur Helminthenfauna der Schafe in Syrien[END_REF] Average II). When considering the total fauna and prevalence of species in sites with high prevalence of Marshallagia (> 50 % hosts), most of the other species were highly variable among sites (Table III). The ratios variance to mean of prevalences were low for Marshallagia and T. probolurus and were high for all the other species or for Nematodirus genus. On the UPGMA performed on the prevalences of parasites in abomasa (Fig. 2) an Eurasian group appears (Syria, Turkey, Kazakhstan, Kashmir and Mongolia). Two groups are climatically driven: one group is in relation to the subhumid climate (Morocco and León 4 in Spain) and another group corresponds to arid climate (León 1 to 3 in Spain and Batna in Algeria).

RELATIONSHIP BETWEEN CLIMATE AND PRESENCE OF MARSHALLAGIA AND OTHER NEMATODE SPECIES

The significant relations between prevalence and climate indicators were: Marshallagia with rainfall (rs = -0.78; n = 16), Martonne index (rs = -0.54) and DS (rs = -0.60), T. circumcincta (Martonne index rs = 0.72, DS rs = 0.79; n = 16), T. axei (rainfall rs = 0.77, DS rs = 0.53; n = 16), and Nematodirus (rainfall rs = 0.73; n = 9). Rainfall was the best indicator for prevalence of Marshallagia and other nematode species. A significant linear regression was established between the prevalence of Marshallagia and rainfall (Fig. 3). Contrasting to most other nematode species, lower rainfall was associated to higher Marshallagia prevalence of infection.

DISCUSSION

M aximum co-speciation has been considered as the norm, but it must be replaced by a more general model of evolution that includes host switching to understand the geographic distribution of host parasite systems. Host-switching and geographical dispersal of parasites are common phenomena in natural host-parasite systems [START_REF] Hoberg | A macroevolutionary mosaic: episodic host-switching, geographical colonization and diversification in complex host-parasite systems[END_REF]. These events of high importance at an evolutionary scale of time may also play a role at the historical scale particularly for domestic hosts and their parasites. The geographic range of domestic herbivores has expanded enormously, as well as the number of hosts (of the original host species and also other species the parasite was in contact with). It remains however difficult to decipher the role of physical environment (climate included) and geographic location per se in the presence of domestic animals in a region. The goat may well have been domesticated a little earlier than other domestic ruminants. The ancestral wild stock exists, is the wild goat (Capra aegagrus), which is found from Anatolia to Pakistan, in the Neolithics [START_REF] Moutou | Geographical distribution of domestic animals: a historical perspective[END_REF]. It is probable that there were two invasions to Africa, in the 5 th and in the 3 rd millennium BP. Sheep were introduced to the southern Levant as fully domesticated by the end of the 9 th millennium BP [START_REF] Haber | Analyzing the process of domestication: Hagoshrim as a case study[END_REF] and presented similar invasions. Thus the area extending from North Africa to Pakistan and possibly Caucasus (supported by the genetic analysis of wild and domesticated sheep: Hienleder et al., 2002) constitutes the original area of domestic sheep and goats at the end of the Neolithic. The presence of the parasitic nematode Marshallagia corresponds in part to the origin of the small ruminants although it has expanded to Spain and mountain areas of several parts of Europe. It has also clearly expanded geographically through the dissemination of domestic sheep and goats. This parasite has been found in wild Ovis (prevalence 11.5 % similar to domestic sheep, in the data set of Suarez & Cabaret, 1999) or Capra (prevalence 15.5 % in the same data set) but was captured by reindeer, muskoxen, and several species of cervids, among which Caribou. There is thus a contradiction to the relatively limited area of Marshallagia in sheep and goats and its capabilities to invade a wide variety of hosts and climates (Fig. 1A). It is an illustration of the Beijerinck's law "Everything is everywhere, but the environment selects" (cited in Perez del [START_REF] Fernandez | Not everything is everywhere: the distance decay of similarity in a marine host-parasite system[END_REF]: "everywhere" concerns mostly wild ungulates and small ruminants and "environment selects" is particularly true for the nematode infection of worldwide distributed domestic sheep and goats. Climate is clearly a key for the distribution of gastrointestinal nematodes of sheep and goats [START_REF] Walkden-Brown | Ecology of the free-living stages of major trichostrongylid parasites of sheep[END_REF]. Moreover, sheep and goats are present throughout the world and if distribution of parasitic nematodes was only host driven, they should be found everywhere, which is not the situation and particularly for Marshallagia. Rainfalls play a major role and temperature (at least in steppe areas) is less important. More sophisticated climatic indices (Martonne or index of departure from steppe climate) are not more efficient for prediction of nematode prevalence in a site. The relation to rainfalls was negative for Marshallagia and was positive for all other species. In fact the highest prevalence was observed in areas with low rainfall (180 to 385 mm per year). The seasonal dynamics indicates that October-November in Morocco is the period with higher intensities [START_REF] Cabaret | Seasonal changes in the abomasal nematodes of naturally infected ewes in Moulay-Bouazza (Morocco)[END_REF]; a similar trend was noted in Algeria (our unpublished results), in Spain [START_REF]Estudio epidemiológico sobre los nematodos gástricos ovinos de la provincia de León, con especial referencia a Ostertagia spp[END_REF] as well as in Uzbekistan [START_REF] Oripov | Epizootiology of trichostrongylid infections in sheep in Uzbekistan[END_REF]. This means that infection should occur at the end of the dry-season or the very beginning of rainy season. This seasonality corroborates the climatic environment needed for Marshallagia, a relative dryness. The capability to withstand dryness is however limited: there is no development from May to September of Marshallagia in very arid steppe of Uzbekistan (Marchanov et al., 1985). The parasite community, with the exception of Marshallagia was highly variable (variance to mean ratio was high for all other specie that were largely represented) and there was no community that could be identified for steppic climate. This climate is then characterised by a single genera (Marshallagia) which is represented by very few species. In the absence of molecular genetic studies it is yet difficult to understand if there was an expansion with a relatively generalist species of parasite (able to survive in different hosts) or if there was a local genetic differentiation between parasitic isolates.

  Original contribution Parasite, 2011, 18, 261-269 THE STEPPE SPECIES OF GASTROINTESTINAL NEMATODES OF SMALL RUMINANTS, WITH A FOCUS ON MARSHALLAGIA: CLIMATE AS A KEY DETERMINANT MERADI S.*, BENTOUNSI B.**, ZOUYED I.** & CABARET J.*** Summary:

  Fig. 1. -Geographic distribution of the nematode Marshallagia marshalli in wild ungulates (A) and domestic sheep and goats (B) established on records from 1930 to 2010.

  -Proportions within the community of nematodes species of sheep and goats based on necropsies (abomasum).

Fig. 2 .

 2 Fig. 2. -Prevalence of nematode species located in abomasum: data of 16 surveys in steppe and cluster analysis using UPGMA and Spearman correlation coefficient.

Fig. 3 .

 3 Fig. 3. -Linear relationship between prevalence of the nematode Marshallagia marshalli and rainfall in 16 surveys in steppe. (Ln: neperian logarithm, R: regression coefficient and P probability of type 1 error).

:

  LES ESPÈCES DE NÉMATODES GASTRO-INTESTINAUX DES PETITS RUMINANTS DE LA STEPPE, AVEC UNE ATTENTION PARTICULIÈRE POUR MARSHALLAGIA : LE CLIMAT COMME DÉTERMINANT ESSENTIEL Notre objectif est de relier la distribution géographique des nématodes gastro-intestinaux des ruminants aux caractéristiques du climat (et de la végétation) steppique. Nous avons utilisé les données de la littérature et d'autres plus récentes que nous avons acquises.

Nous avons recouru à des indices simples ou sophistiqués pour caractériser le climat. Des analyses de régression ont été utilisées pour estimer les relations entre les nématodes gastro-intestinaux et les facteurs climatiques. La distribution d'un genre (Marshallagia) sur les cinq rencontrés était surtout concentrée dans les zones steppiques, alors que les autres espèces étaient également rencontrées dans d'autres régions. Chez les hôtes sauvages, la distribution était beaucoup plus large, du Spitzberg au Nouveau Monde (territoires du nord du Canada et extrême sud de l'Amérique). Chez les petits ruminants domestiques, la présence de Marshallagia a été recensée de manière plus intense et plus constante dans les zones initiales de domestication ou dans ses diffusions proches (de l'Afrique du Nord au Cachemire ou au Caucase). La distribution de ce parasite était reliée à de faibles pluies, à l'inverse de ce qui était noté pour les autres nématodes. Ce n'est que lors de captures (renne, camélidés sud américains) que ce nématode a élargi son aire de distribution, dans des zones plus froides ou plus sèches. MOTS-CLÉS : petit ruminant, nématode endoparasite, Marshallagia, climat, steppe, capture.

  Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2011183261 Original contribution

	Parasite, 2011, 18, 261-269

Table I .

 I -Prevalence of nematodes groups in sheep and goats based on faecal egg counts.

  ). The prevalence of Marshallagia ranged from 10 (Kazakhstan) to 85 % Original contribution

						Site
	Morocco		Mongolia	Kashmir	Syria 4		Spain
	Moulay	Moulay	Moulay	Province	Province		North west	León	León	León	León
	1	2	3	1	2	Valley	areas	1	2	3	4
	Parasite, 2011, 18, 261-269

Table III

 III It should be noted that it may vary much from different sites in the same country (21 to 81 % in Algeria). The importance of other nematodes characterized different sites with the high proportion of H. contortus in Kashmir valley and in Mongolia, of T. vitrinus in Syria 4, of Trichostrongylus axei in Morocco and León (Table

. -Prevalences of nematode species in sheep and goats: necropsies of all gastrointestinal-tract in highly Marshallagia infected sites. Original contribution Parasite, 2011, 18, 261-269 (Algeria).

ACKNOWLEDGEMENTS

W e are grateful to the University of Batna (Algeria) for funding two stays in France (INRA) to one of us (S.M) that permitted the planning of investigations and analyses of the data.


B Langlois -

C Blouin

S Chaffaux

Analysis of several factors of variation of gestation loss in breeding mares

Keywords: horse, gestation loss, factor of variation, logistic regression Implications

The files for ultrasound diagnosis of gestating mares belonging to the French equine herd recorded for 3 consecutive years were joined with the files for foal birth of these same mares, allowing the statistical analysis of factors of pregnancy loss. For 28 872 positive diagnoses of gestation, 2898 losses were recorded, that is, a global rate of gestation interruption of 9.12%. The etiology of these interruptions is mainly extrinsic: the year and month of insemination, as well as region for climatic reasons. The intrinsic causes that are implicated are breed of the father (heavy breeds except the hypermetric ones lose fewer pregnancies than warmblooded breeds), age of the mother (losses are lower in mares of 7 to 10 years of age) and status (mares with foals have fewer pregnancy losses than mares not having foaled the previous year), as well as fetuses with consanguinity (when this increases, the pregnancy losses increase as well). However, the additive genetic effect is extremely low; it corresponds to heritability below 5% and few effects of the environment, common to the offspring of the same mare, were identified. This therefore gives little hope of being able to select against the 'gestation loss' trait.

Introduction

It was shown by an economical inquiry conducted by the 'REFErences' network in France (2010) that the low economical performance of horse breeding is in great part explained by the low performances in reproduction. We analyzed some factors of variation of numerical productivity [START_REF] Langlois | Statistical analysis of some factors affecting the number of horse births in France[END_REF]. Here, because of the national service of gestation diagnosis by ultrasound developed by the national studs, we have the possibility (unique in the word) to study some factors of pregnancy losses on a very large scale with the same statistical methods. This is the objective of this paper.

Joining mating files with those of birth recordings has already shown [START_REF] Langlois | Statistical analysis of some factors affecting the number of horse births in France[END_REF][START_REF] Sairanen | Effects of inbreeding and other genetic components on equine fertility[END_REF]) variation factors for the numerical productivity of breeding mares on a large scale. However, this numerical productivity is a global zootechnical parameter that results from fertilization, rate of early embryonic resorption, abortion rate, stillbirth rate and even sometimes the breeder's choice to not record a foal's birth. In France, the National studs have set up a service for gestation diagnosis using transrectal ultrasound for all breeds. Immediately after being developed, this method was used by pioneers to measure the incidence of gestation loss on the mare (Chevalier and [START_REF] Palmer | Ultrasonic echography in the mare[END_REF]. These authors observed 69 gestation losses for 1295 positive ultrasounds, that is, a 5.3% loss.

A first ultrasound is performed between 14 and 20 days following insemination. Then, a positive result is as a rule verified before endometrial cupula formation (43 to 45 days -E-mail: bertrand.langlois@jouy.inra.fr after fertilization). It is therefore possible to evaluate gestation losses that occur between blastocyte immobilization and birth recordings using the files from the ultrasound results and foal birth recordings, which represent a large number of fertilizations. In this paper, we will present the variation factors for these gestation losses.

Material and methods

Material

The ultrasound diagnosis of the gestation service of the Haras Nationaux is available all over France. For the 2005, 2006 and 2007 breeding seasons, 31 770 mares were declared pregnant by ultrasound, but only 28 872 had a foal declared at birth. Some breeding mares were fertilized several times during these three breeding seasons. Indeed, there were only 23 017 different mares, that is, 1.4 observations on average per mare.

Methods

For each mare declared pregnant during a particular year, a bimodal variable was created that was equal to 1 if the positive ultrasound diagnosis was followed by a birth record and 0 if no birth record was made.

The study of the factors of variation was performed by logistic regression (SAS Institute, 2003), which is the appropriate method for bimodal variables. A certain number of simplified models were previously applied to regroup the effects and remove the non-significant effects from the final analysis. This model was applied by adding a random effect for the mare using the Genmod procedure (SAS Institute, 2003), to obtain an idea of the mother's effect and its repeatability, as well as the father's effect in order to evaluate heritability.

The fixed effects examined were the following: The father's breed: a total of five warm-blooded breeds and eight cold-blooded breeds were distinguished. The following are the warm-blooded breeds: Thoroughbreds (PS), Arab (AR), Anglo-Arab (AA), French trotter (TF) and French saddle breeds (SF). The following are the draught breeds: Percheron (PER), Boulonnais (BOU), Breton (BRE), Normandy Cob (CBN), Comtois (COM), Ardennais (ARD), Trait du Nord (TDN) and Auxois (AUX).

Preliminary analyses allowed the following regroupings: for draught horses, the heavy breeds (PER 1 BOU), averagesized breeds (BRE 1 CBN), light breeds (COM) and all Ardennais type (ARD 1 TDN 1 AUX). For warm-blooded breeds, we did not find it necessary to distinguish between AR and AA for the reproductive characters.

We therefore obtained eight groups of breeds for the fathers for which the most numerous BRE 1 CBN served as a reference.

The mother's breed: in order to avoid multiplying the levels, only two modalities were examined; the mother's breed was identical to that of the father or it was different. As the advantage of crossbreeding did not appear significant, it was abandoned. The mother's status: this includes whether or not the mare has a foal with her. Mares with foals served as references.

The mother's age: the seven following categories were retained: younger or equal to 2 years; between 3 and 4 years; 5 to 6 years; 7 to 10 years was the reference; 11 to 15 years; 16 to 20 years; 21 years or older.

The last mating month: January to February; March; April; May was the reference; June; July to December; that is, six levels.

1. Year of mating: 2005 was taken as the reference; 2006 and 2007, that is, three levels. 2. Inbreeding: that of the mother and of the foal to be born was calculated using the PEDIG software [START_REF] Boichard | Pedig: a Fortran package for pedigree analysis suited for large populations[END_REF]. The equivalent number of completely known generations for birth year 2008 varied from 2.5 for French saddle to 8.9 for Thoroughbred for warm-blooded breed and from 4.6 for Ardennes to 7.4 for Comtois for coldblooded breeds. This is a relatively profound information that shows that close inbreeding is taken into account in any case even if as usual remote inbreeding could mostly escape. Four categories were made according to the consanguinity value f: f 5 0 reference; f . 0 and <0.0625; f . 0.0625 and <0.125; f . 0.125. Only one coefficient for consanguinity of the foal to be born was significant.

The inbreeding effect of the mother was abandoned. 3. The region effect: this was considered on the 95department level, 22 regions or a logical regrouping of these regions as a function of the preliminary results observed on the department level. Here is how we proposed the 10 following regions: For the presentation of the results, we chose to determine the odds ratios (OR), which were calculated by determining the exponential of the logistical regression coefficients. They express the increase or decrease of chances to obtain a specific value of the bimodal variable for a certain level taken as the reference. For the variation factors with positive effects, OR . 1. If this effect is negative OR , 1 and the reference level will be OR 5 1.

(i) Alsace 1 Champagne Ardennes 1 Lorraine 1 Franche comte (ii) Brittany (iii)
For the study of variation factors, only the results corresponding to the complete model are presented:

The declaration of a product 5 effect of the father's breed (8 levels) 5 effect of the mother's status (2) 1 the effect of the mother's age (7) 1 the effect of the mating month (6) 1 year effect (3) 1 inbreeding effect of the product to be born (4) 1 region effect (95/10) 1 residual effect.

The adjustment of the effects was obtained using the PROC LOGISTIC procedure of SAS Institute (2003).

Results

Globally, gestation losses, as we have defined them, were equal to 2898 for a total number of mares of 31 770, that is, 9.12%.

Effect of the father's breed When warm-blooded breeds and cold-blooded breeds were considered together, the products of warm-blooded breeds were more often lost (OR 5 0.925) before the birth recordings than were the products of draught breeds (OR 5 1). This, however, cannot be generalized for all heavy breeds. Boulonnais and Percheron breeds and the Ardennes-type breeds in particular had significantly lower results than the other heavy breeds. Among warm-blooded breeds, the French trotter seemed to have a better performance but equal to that of the Comtois. Arab and Anglo-Arab, Thoroughbred and French saddle breeds cannot be distinguished from each other. The Breton and Normandy cob are significantly above the other breeds. They had fewer gestation losses after a positive ultrasound diagnosis (Figure 1).

The status of the mother We observed a highly significant advantage of mares with foals as compared with mares without foals: pregnant mares with foals at feet had a better chance of bringing their pregnancy to term than mares without foals (Figure 2).

Effect of the age of the mothers If age classes >21 years are excluded, as they are poorly estimated and subject to selection bias, we observed that maintaining gestation reaches a maximum for mares whose age is between 7 and 10 years. Gestation losses were more frequent before or after these ages (Figure 3).

Effect of breeding month

If January and February are excluded, which are poorly estimated because of the lack of data and for which a too high value necessitates shrinking the values of the other months, a decrease in the breeding animals' success was observed, in terms of gestation success. This depends on whether the fertilization occurred in March or later in the season (Figure 4).

Year effect

During 2006, there were highly significant fewer gestation losses than the years 2005 and 2007 (Figure 5).

Effect of inbreeding on foal birth

The chances for survival of a foal decreased when the consanguinity coefficient was .0.0625, whereas the consanguinity of the mare did not seem to have an effect. Gestation losses, which were still low at the beginning, become highly significant when f was .0.125 (Figure 6).

Department and region effect

A department-by-department analysis shows few significant differences when the Manche (50) was the reference. Only two of them, departments 72 and 43, had significantly higher results and 10 had significantly lower results (05, 07, 09, 20, 37, 42, 52, 60, 89 and 94).

However, grouping the departments into 10 large regions, as indicated above, gave greater cohesion to the results. The East and the Mountain regions had significantly lower performances when compared with the larger reference zone, which included Normandy and the Pays de Loire (Figure 7).

Estimation of the repeatability of the results per mare and per paternal component

The results obtained for the fixed effects by the PROC GENMOD procedure of SAS Institute (2003), when the random mother or father effects are added, were identical. They will only be considered under the angle of variance for which they provide access.

Adding a random component because of the mother or father allowed the estimation of an intra-class mother correlation of 3.7%, whereas the intra-class correlation of the father was evaluated at 1.3%. In the first case, 23 017 breeding mares with 1 to 3 observations were available and in the second 1020 stallions with 1 to 302 observations. These two estimations were coherent and do not leave hope that selection will reduce gestation losses. This result was a common result as the traits associated with fertility have themselves already undergone a very strong automatic selection pressure and only conserve very little additive genetic variability. They therefore have a low heritability.

Discussion

The overall rate of loss measured here (9.1%) was comparable to that reported by other authors: 7.2% in Thoroughbreds in England [START_REF] Morris | Reproductive efficiency of intensively managed Thoroughbred mares in Newmarket[END_REF][START_REF] Allen | Reproductive efficiency of Flat race and national Hunt Thoroughbred mares and stallions in England[END_REF] or in Australia [START_REF] Bruck | Reproductive performance of Thoroughbred mares on six commercial stud farms[END_REF] and 8.6% in different breeds in a review article [START_REF] Vanderwall | Early embryonic loss in the mare[END_REF]. However, it was lower than those observed for Thoroughbreds in India, 19% [START_REF] Sharma | Reproductive efficiency of Thoroughbred mares under Indian subtropical conditions: a retrospective survey over 7 years[END_REF] or 12% in South Korea [START_REF] Yang | Factors concerning early embryonic death in Thoroughbred mares in South Korea[END_REF].

The comparison of the observed effects with those published in 2004 [START_REF] Langlois | Statistical analysis of some factors affecting the number of horse births in France[END_REF] for numerical productivity of breeding mares revealed similarities and differences. In the previous study, draught horses had a lower numerical productivity than that of Thoroughbreds. However, here, they had a significantly lower early gestation loss. The lower losses of the Breton and Normandy Cob, however, did not have a notable effect on their numerical productivity. In addition, the high rates of gestation losses of the Boulonnais and Percheron and in particular the group of Ardennes, Trait du Nord and Auxois had little incidence on the overall productivity results as measured in the first study. It is important to recall that these effects, even though very marked, had an effect on less than 10% of the overall result and that they may be compensated for by a higher fertility rate. It is noteworthy that in unusually developed breeds, birthing difficulties are suggested to explain their greater gestation losses. Indeed, a partial epidemiological study on 185 gestations [START_REF] Chaffaux | Cre ´ation d'un re ´seau d'e ´pide ´miosurveillance des avortements, de la mortinatalite ´et de la mortalite ´ne ´onatale infectieux et contagieux chez les e ´quide ´s, en France. Bilan des deux premie `res anne ´es d'activite ´[END_REF]; Figure 8) showed the great influence of stillbirth on fetal losses.

For warm-blooded breeds, the lower rate of gestation loss in the French trotter confirms its overall greater global efficacy; however, the differences in numerical productivity observed in the first study between Thoroughbreds and saddle breeds end here for gestation losses.

The advantage of mares with foals over mares without foals was, however, of the same order in both studies (OR no. 1.35 as compared with 1.30 here). This confirms that does not have an influence on the rate of loss of gestation products [START_REF] Vanderwall | Early embryonic loss in the mare[END_REF]. This may be explained by the fact that the mares without foals include in fact virgin mares, a priori fertile, and mares that remained not gestating during the preceding season, a priori less fertile or even sterile. However, [START_REF] Allen | Reproductive efficiency of Flat race and national Hunt Thoroughbred mares and stallions in England[END_REF] did not find this difference for English Thoroughbred mares with foals and without foals.

The effect of the age of the mother on gestation loss was not studied with the same precision as that in the first study on numerical productivity, forcing larger classes with higher standard deviations. However, the existence of a certain parallelism remains. Whereas numerical productivity reaches a maximum between 4 and 6 years of age, for gestation loss the minimum is reached later near 7 years with a higher persistence. Fertility is therefore first affected by the age of the mare; the rate of fetal loss is affected later. This age effect of the breeding mare on the rate of gestation loss was observed in an identical manner by all authors [START_REF] Allen | Reproductive efficiency of Flat race and national Hunt Thoroughbred mares and stallions in England[END_REF][START_REF] Yang | Factors concerning early embryonic death in Thoroughbred mares in South Korea[END_REF][START_REF] Vanderwall | Early embryonic loss in the mare[END_REF][START_REF] Sharma | Reproductive efficiency of Thoroughbred mares under Indian subtropical conditions: a retrospective survey over 7 years[END_REF]. During the genital life of a mare, her stock of oocytes loses their capacity to be fertilized more rapidly than the ageing of their oviducts and uterus. This explains why the rate of fertilization decreases more rapidly, like the numerical productivity, than the increase in gestation loss (Ball et al., 1987).

Factors of variation of gestation loss in mares

The season effect of reproduction is parallel both studies. Here we may suppose that the hormonal state of the mare induced by the day/night rhythm influences both the fertilizing conditions and maintaining of gestation. The same effect was observed in South Korea by [START_REF] Yang | Factors concerning early embryonic death in Thoroughbred mares in South Korea[END_REF]. However, in India where the day/night variations are less important, this season effect was not observed; the rate of loss was identical all throughout the year [START_REF] Sharma | Reproductive efficiency of Thoroughbred mares under Indian subtropical conditions: a retrospective survey over 7 years[END_REF]. One must, however, remember that at the beginning of the season a larger number of mares that in the previous year were sterile were inseminated and presented higher gestation loss rates than the others, which may partly explain these lower results in March to April.

The year effect, limited here to 3 years, did not reveal any general tendency to improvement, nor deterioration as in the previous study. This confirms that the loss during gestation is independent of technical improvements. They are permanent on the contrary to numerical productivity, which benefitted from the increasing improvements in ultrasound scan and artificial insemination during the period. Indeed, the increase in fertility rate observed was attributed to an increased technicality of reproduction. However, the important effect of the year 2006 marked by a significantly lower rate of gestation loss shows that the climate, feeding and/or epidemiology can have a high influence on gestation loss.

It is this type of an effect that was sought for in the regional differences of gestation loss rate. The department scale shows little differences. Contrarily as we expected, we could therefore not show the regions with endemic infectious diseases. However, it seems that the Large East zone and the Mountain zones with a harsh climate, with a less rich feeding in winter show significantly higher losses than in the oceanic and Mediterranean zones.

The effect of foal consanguinity shown in previous studies on numerical productivity was confirmed here on the rate of gestation loss. However, the additive genetic effect was extremely low, corresponding to heritability below 5%. It is in fact not possible to identify many environmental effects common to the same mare because the mare's component of variance is of the same order of magnitude as the sire's component.

These results show that it is essentially the environmental factors that are at the origin of the loss of fertility products. Even though the genetic effects are involved as the consanguinity effect shows, it is mainly at the level of allele interactions expressed by dominance and epistasis and probably not at the level of additive effects. We can therefore obtain positive effects by crossbreeding, even though here they did not appear to be significant, when we studied the mother's breed identical to or different from the father's breed. However, nothing should be expected from selection.

Conclusion

In this study, we show that between the moment when a mare was diagnosed as being pregnant by ultrasound and the moment of birth declaration , 10% of the products were lost. The factors that are implicated in these losses are essentially extrinsic; they depend mainly on the environment. They are mainly region, month of insemination and year. Certain effects are, however, intrinsic, being associated with the mother: her age and if she is with or without a foal, the breed of the father and fetus (its consanguinity). We may not, however, determine strong genetic effects allowing selection on this trait. If these genetic effects do exist, it is via gene interactions that are by dominance or epistasy, therefore explaining the negative effect of consanguinity. A distinction between these different causes of gestation losses (embryo mortality, miscarriage, stillbirth, neonatal death) may help explain this problem.

Figure 1

 1 Figure 1 The effects of the father's breed expressed as an odds ratio. PER 1 BOUL 5 Percheron 1 Boulonnais; BRE 1 CBN 5 Breton 1 Cob Normand; ARD et al. 5 Ardennes 1 Trait du Nord 1 Auxois; TH 5 Thoroughbred; AA 1 AR 5 Anglo-Arab 1 Arab; SF 5 Selle Franc ¸ais (French Saddle); TF 5 Trotteur Franc ¸ais (French Trotter).

Figure 2

 2 Figure2Effect of mare's status, with or without foals, expressed as an odds ratio.

Figure 3

 3 Figure 3 Effects of age classes of the mother expressed as an odds ratio. <2y 5 2 years old and less; 3 to 4y 5 3 to 4 years old; 5 to 6y (ref) 5 5 to 6 years old (the reference); 7 to 10y 5 7 to 10 years old; 11 to 15y 5 11 to 15 years old; 16 to 20y 5 16 to 20 years old; .20y 5 older than 20 years.

Figure 4

 4 Figure 4 Effects of season of the last mating expressed as an odds ratio.

Figure 5

 5 Figure 5 Effect of year expressed as an odds ratio.

Figure 6

 6 Figure 6 Effect of consanguinity level expressed as an odds ratio. Class 1 5 f 5 0; class 2 5 0 , f < 0.0625; class 3 5 0.0625 , f < 0.125; class 4 5 f . 0.125.

Figure 7

 7 Figure 7 Effect of the 10 regions on rate of early gestation losses. (In yellow the two regions having significantly higher losses than the High and Low Normandy region and Pays de la Loire.)

Figure 8

 8 Figure 8 Fetal losses according to the month of gestation (n 5 185).

  Basse Normandy 1 Haute Normandy 1 Pays de Loire (the reference) (iv) Ile de France 1 Nord pas de Calais 1 Picardie (v) Re ´gion Centre 1 Limousin 1 Charente Poitou (vi) Aquitaine 1 Midi Pyre ´ne ´es without the mountain departments (09, 64, 65) and Aveyron (12) (vii) Auvergne and Aveyron (12) and Loze `re (48) (viii) Bourgogne 1 Rho ˆne Alpes without the mountain departments (07, 38, 73 and 74) (ix) Corse 1 Languedoc Roussillon 1 Provence Alpes

	Cote d'Azur without the mountain departments
	(05, 48 and 66)
	(x) A 10th fictive region was created with the following
	mountain departments: 05, 07, 09, 38, 64, 65, 66,
	73, 74.

Acknowledgment

The authors are grateful to Dr Be ´ne ´dicte Ferry-Abitbol for providing them the list of the gestating mares observed by ultrasound rectal examination from 2005 to 2007.