Herve ´fritz
email: herve.fritz@univ-lyonl.fr

Michel Loreau

Simon Chamaille ´-Jammes

Marion Valeix

Jean Clobert

H Fritz

Á M Loreau

Á S Chamaille´-Jammes

A food web perspective on large herbivore community limitation

The exceptional diversity of large mammals in African savannas provides an ideal opportunity to explore the relative importance of top-down and bottom-up controls of large terrestrial herbivore communities. Recent work has emphasized the role of herbivore and carnivore body size in shaping these trophic relationships. However, the lack of across-ecosystem comparisons using a common methodology prohibits general conclusions. Here we used published data on primary production, herbivore and carnivore densities and diets to estimate the consumption fluxes between three trophic levels in four African savanna ecosystems. Our food web approach suggests that the body size distribution within and across trophic levels has a strong influence on the strength of top-down control of herbivores by carnivores and on consumption fluxes within ecosystems, as predicted by theoretical food web models. We generalize findings from the Serengeti ecosystem that suggest herbivore species below 150 kg are more likely to be limited by predation. We also emphasize the key functional role played by the largest species at each trophic level. The abundance of the largest herbivore species largely governs the consumption of primary production in resident communities. Similarly, predator guilds in which the largest carnivore species represent a larger share of carnivore biomass are likely to exert a stronger top-down impact on herbivores. Our study shows how a food web approach allows integrating current knowledge and offers a powerful framework to better understand the functioning of ecosystems.

Understanding the relative importance of top-down and bottom-up controls in terrestrial ecosystems is a challenging issue in ecology, in particular in ecosystems dominated by large mammals [START_REF] Halaj | Terrestrial trophic cascades: how much do they trickle? Á[END_REF]Wise 2001, Shurin et al. 2002). Although strong trophic cascades may occur when vertebrate endotherm predators are extirpated or introduced [START_REF] Borer | What determines the strength of a trophic cascade?[END_REF], as exemplified in some Northern American systems [START_REF] Mclaren | Wolves, moose, and tree rings on Isle Royale[END_REF]Peterson 1994, Ripple and[START_REF] Ripple | Wolves and the ecology of fear: can predation risk structure ecosystems?[END_REF] and suggested in Australia [START_REF] Letnic | Are dingoes a trophic regulator in arid Australia? A comparison of mammal communities on either side of the dingo fence[END_REF], the lack of comparative food web analyses of large diverse mammalian communities precludes broad conclusions [START_REF] Duffy | The functional role of biodiversity in ecosystems: incorporating trophic complexity[END_REF]. African savannas are terrestrial ecosystems in which there is still a high diversity of large mammalian herbivore and carnivore species (hereafter large herbivores and large carnivores respectively; [START_REF] Fritz | Large herbivores across biomes[END_REF], contrary to most temperate systems where large predators have been extirpated by humans. This unique feature might make African savannas different from other terrestrial systems regarding the relative importance of bottom-up and topdown processes in limiting large vertebrate communities. The effect of large carnivores on ungulate populations in species-rich communities was brought to attention several decades ago [START_REF] Smuts | Interrelations between predators, prey, and their environment[END_REF]. Many population studies, however, have suggested bottom-up control of large herbivores in African savannas [START_REF] Owen-Smith | Demography of a large herbivore, the greater kudu Tragelaphus strepsiceros, in relation to rainfall[END_REF][START_REF] Mduma | Food regulates the Serengeti wildebeest: a 40-year record[END_REF], supported by analyses at the community level showing that large ungulate biomass was directly related to primary production (Coe et al. 1976, Fritz and[START_REF] Fritz | On the carrying capacity for large ungulates of African savanna ecosystems[END_REF]. A positive relationship between primary and secondary production is also observed across terrestrial ecosystems [START_REF] Mcnaughton | Ecosystem-level patterns of primary productivity and herbivory in terrestrial habitats[END_REF]). However, recent population studies suggest predation limits some large herbivore populations [START_REF] Sinclair | Patterns of predation in a diverse predatorÁprey system[END_REF][START_REF] Grange | What limits the Serengeti zebra population?[END_REF][START_REF] Owen-Smith | Manifold interactive influence on the population dynamics of a multispecies ungulate assemblage[END_REF].

Population limitation mechanisms may differ between species in a community. In particular, body size is likely to play a major role in setting the relative roles of predation and resource availability at the population level. Models show that herbivore-plant size ratios may influence the strength of trophic cascades [START_REF] Shurin | The strength of trophic cascades across ecosystems: predictions from allometry and energetics[END_REF], and that the distribution of body sizes within communities may determine the relative importance of top-down and bottom-up controls in entire food webs [START_REF] Loeuille | Evolutionary emergence of size-structured food webs[END_REF]. A natural experiment of predator removal in an East African savanna recently underlined the role of predation in the regulation of small-and medium-sized ungulate populations [START_REF] Sinclair | Patterns of predation in a diverse predatorÁprey system[END_REF], suggesting a potential for strong trophic cascades in this system. In multispecies prey communities, a predator may also strongly regulate its primary prey population if there are secondary prey that provide enough energy to subsidize it in times when primary prey decrease in abundance [START_REF] Sinclair | Patterns of predation in a diverse predatorÁprey system[END_REF]. [START_REF] Sinclair | Patterns of predation in a diverse predatorÁprey system[END_REF] also suggested that the diet breath, body size and species diversity of predators may enhance the role of predation in limiting some populations, in line with theoretical and empirical work conducted in widely different systems [START_REF] The ´bault | Food-web constraints on biodiversityÁecosystem functioning relationships[END_REF]Loreau 2003, Jiang and[START_REF] Jiang | Predator diet breadth influences the relative importance of bottom-up and top-down control of prey biomass and diversity[END_REF]. [START_REF] Hopcraft | Herbivore, resources and risks: alternating regulation along primary environmental gradients in savannas[END_REF] recently reviewed the literature on African savannas and offered a general framework to predict the relative contribution of bottom-up and top-down controls on herbivores across environmental gradients. They argued that the functioning of savannas is primarily determined by a) vegetation quality and productivity that directly influence the differential performance of herbivores of different body sizes, and b) how carnivores of different body sizes partition their food niches along a herbivore body size gradient. However, the paucity of across-ecosystem comparisons using common standardized methods creates an important gap in our ability to assess this framework.

Here we develop a food web approach (i.e. a quantification of biomass consumption across species or trophic levels, hereafter referred to as consumption fluxes) to assess the relative importance of top-down and bottom-up processes in controlling ungulate populations in four species-rich African savanna ecosystems. Our approach does not aim to describe the dynamics of the system, as the relative strength of interactions may change over time concordant with changes in the relative abundances of prey and predator species, but to quantify consumption fluxes between species and trophic levels to assess whether predation was likely to limit large herbivore populations and thus to provide insights on the factors governing food web structure. Comparative ecosystem studies have proved useful for investigating the strength of trophic cascades and their possible sources of variation [START_REF] Halaj | Terrestrial trophic cascades: how much do they trickle? Á[END_REF][START_REF] Shurin | A cross-ecosystem comparison of the strength of trophic cascades[END_REF][START_REF] Borer | What determines the strength of a trophic cascade?[END_REF]). Here we use ecosystems that show contrasting consumption fluxes between primary producers and the various large herbivore species, as well as diverse predation pressures (Fig. 1). We quantify herbivory and predation in ecosystems with a high biomass of large herbivores, for which data are scarce [START_REF] Schmitz | Trophic cascades in terrestrial ecosystems: a review of the effects of carnivore removals on plants[END_REF][START_REF] Halaj | Terrestrial trophic cascades: how much do they trickle? Á[END_REF][START_REF] Shurin | A cross-ecosystem comparison of the strength of trophic cascades[END_REF][START_REF] Borer | What determines the strength of a trophic cascade?[END_REF]. By taking the complexity of trophic relationships into account and applying the same methodology to different ecosystems, we aim to better understand the processes regulating trophic cascades, and particularly the role of body size in these processes. We expect resident herbivore communities composed mostly of small species (sensu Sinclair et al. 2003: B150 kg) to be more influenced by top-down processes than those dominated by large herbivores. This should also result in the former showing lower herbivory pressure.

Materials and methods

Study systems

The four studied savanna ecosystems Á Hwange, Kruger, Ngorongoro and Serengeti Á are all protected areas. The four ecosystems differ in soil nutrient availability (sensu [START_REF] Bell | The effect of soil nutrient availability on the community structure in African ecosystems[END_REF]: Hwange National Park (Zimbabwe; 19.18S, 26.68E) has poor soils, Kruger National Park (South Africa; 24.08S, 31.58E) has medium-poor soils, and Serengeti National Park and Ngorongoro Crater National Park (Tanzania, respectively: 2.58S, 34.68E and 3.18S, 35.48E) have rich soils. We focused on the northern section of the Serengeti and on the Main Camp section of Hwange National Park, for which we had data on carnivore diets. We considered that migratory ungulates are present 6 months in a year in the northern section of the Serengeti [START_REF] Fryxell | Aggregation and migration by grazing ungulates in relation to resources and predators[END_REF].

These systems also correspond to different profiles in the biomass distribution across large herbivore body sizes. Hwange is dominated by megaherbivores (sensu Owen-Smith 1988, body weight 1000 kg), mostly elephants Loxodonta africana, the Serengeti and Ngorongoro by medium-to large-sized species (150Á300 kg), while the Kruger is more balanced (Fig. 1). An additional interest of using the Serengeti is that we can compare the results of our food web approach and our subsequent predictions on the relative role of predation with those of an earlier natural predator removal experiment [START_REF] Sinclair | Patterns of predation in a diverse predatorÁprey system[END_REF]. For this comparison, we used the northern section of the Serengeti and distinguished resident and migratory ungulates [START_REF] Sinclair | Patterns of predation in a diverse predatorÁprey system[END_REF] because migration is an efficient anti-predator strategy that has a considerable impact on the trophic fluxes in this ecosystem [START_REF] Fryxell | Aggregation and migration by grazing ungulates in relation to resources and predators[END_REF]. Predator species are the same in the four systems, but their abundance and consumption impact on herbivores differ substantially (Fig. 1).

Data and analyses

We used published data on primary production, herbivore and carnivore densities and diets for the Kruger, Serengeti and Ngorongoro [START_REF] Kruuk | The spotted hyena: a study of predation and social behaviour[END_REF][START_REF] Schaller | The Serengeti lion: a study of predatorÁprey relations[END_REF][START_REF] Sinclair | Serengeti: dynamics of an ecosystem[END_REF][START_REF] Homewood | Maasailand ecology: pastoral development and wildlife conservation in Ngorongoro, Tanzania[END_REF][START_REF] Scholes | An African savanna. Synthesis of the Nylsvley study[END_REF][START_REF] Caro | Cheetahs of the Serengeti plains: group living in an asocial species[END_REF][START_REF] Sinclair | Serengeti II: dynamics, management and conservation of an ecosystem[END_REF][START_REF] Boone | Addressing management questions for Ngorongoro Conservation Area, Tanzania, using the Savanna modelling system[END_REF][START_REF] Du Toit | The Kruger experience: ecology and management of savanna heterogeneity[END_REF][START_REF] Estes | Downward trends in Ngorongoro Crater ungulate populations 1986Á2005: conservation concerns and need for ecological research[END_REF], and unpublished data for Hwange (Drouet-Hoguet, Rasmussen, Fritz, Loveridge and Macdonald pers. comm.).

We converted population density into biomass for the various herbivore species using body mass data from [START_REF] Coe | Biomass and production of large African herbivores in relation to rainfall and primary production[END_REF], which were completed by [START_REF] Cumming | Ungulate community structure and ecological processes: body size, hoof area and trampling in African Savannas[END_REF]. African herbivore communities cover a wide range of body sizes, ranging from steenbok Raphicerus campestris (female adult body mass 011 kg) to elephant (female adult body mass 02275 kg). Body size determines many physiological and ecological attributes [START_REF] Peters | The ecological implications of body size[END_REF]. In particular, metabolic needs scale allometrically with body mass, hence we estimated herbivore dry matter daily food intake from allometric studies [START_REF] Illius | Modelling the nutritional ecology of ungulate herbivores: evolution of body size and competitive interactions[END_REF]. Besides, age at first reproduction and fecundity scale inversely with body size, hence we used these life history traits or [START_REF] Sinclair | Mammal populations: fluctuation, regulation, life history theory and their implications for conservation[END_REF] allometric equation to calculate the maximum annual rate of population increase for each herbivore species assuming no mortality.

Daily food requirements for each carnivore species (in kg of fresh meat) was estimated using published values from a recent review [START_REF] Carbone | The costs of carnivory[END_REF]). We used ecosystem-specific data on carnivore diets to estimate at each system the contribution of each prey species to the annual consumption of each carnivore species. For each herbivore species, we then converted the biomass consumed by all carnivores in a year into a number of individuals consumed, and calculated annual predation rate as the proportion of individuals consumed by carnivores in the population. Lastly, we estimated population growth rates of herbivores after predation as the difference between the maximum annual rate of population increase and the annual predation rate.

To estimate the aggregate consumption fluxes between trophic levels in each ecosystem, we calculated the fraction of primary production consumed by all large herbivores, distinguishing between three body size classes (mesoherbivores B150 kg, mesoherbivores ]150 kg, and megaherbivores), and the fraction of herbivore biomass consumed by all carnivores. Statistical analyses of annual predation rates used log-transformed data to conform to the requirements of parametric statistics.

Results

Consumption fluxes within the studied ecosystems are presented in Fig. 1. Annual predation rate decreased significantly with increasing prey body size (F 1,44 0 10.95, p00.002, r 2 00.60), i.e. smaller-sized herbivores were more heavily impacted by predation (Fig. 2), and there was no significant system effect (F 4,44 00.67, p00.62). The expected population growth rates of small-to mediumsized herbivores, after accounting for predation, were negative (Fig. 2). Based on these estimates, predators were likely, not only to limit, but even to reduce small herbivore populations.

The Serengeti resident system and the Kruger system both had a greater consumption of herbivores by carnivores than of plants by herbivores, whereas the reverse was true for Ngorongoro and Hwange (Fig. 3). The predator:prey biomass ratio (excluding elephants) was similar between Hwange and Ngorongoro (0.008 and 0.01, respectively), and was at least twice lower than in the Serengeti resident system and the Kruger (0.03 and 0.02, respectively).

Hwange was the only system where the contribution of elephants changed the predator:prey ratio significantly; its predator:prey ratio including elephants (0.002) was close to the lowest ratio observed in the Serengeti migratory system (0.001).

The overall impact of predation on prey in each system depended on its predator:prey biomass ratio since the rank correlation between the average predation rate and the predator:prey biomass ratio was significant (n 05, r s 00.9 and r s 01, both pB0.05, with and without elephants in the predator:prey ratio calculation, respectively). This conclusion did not hold for the predator:prey density ratio (both p0.05), which suggests that the body mass of the dominant carnivores played a greater role than their density. Systems where lions Panthera leo comprised most of the carnivore biomass exhibited greater predation pressure on most herbivore body size classes (Fig. 4), and were characterised by greater predation pressure on large herbivores between 700 and 900 kg, which had an estimated growth rate close to zero (Fig. 4).

Discussion

Our food web approach suggests that predation could be limiting in most systems for small-and medium-sized ungulate species, thus supporting and extending previous findings by [START_REF] Sinclair | Patterns of predation in a diverse predatorÁprey system[END_REF] (although in Kruger the high abundance of lions also subjects large preys to high predation pressure Á see also Owen-Smith and Mills 2008).

Figure 2. Maximum annual rates of population increase, estimated annual predation rates averaged across ecosystems (mean 9 SD), and estimated annual population growth rates averaged across ecosystems (mean 9 SD) after accounting for predation for the seven body mass classes of herbivores. Species with body mass below 150 kg appear more likely to be limited by predation, as they exhibited estimated negative or zero growth. a Includes Suids, which have much higher maximum population rate of increase (100%) but also much higher observed annual predation rate (80%).

Figure 3. Percentage of the lower trophic level consumed yearly by carnivores and three herbivore body size classes, calculated as proportions of total herbivore biomass consumed for carnivores, and proportions of total primary production consumed for herbivores. Herbivores are grouped in three classes instead of seven, both for clarity and because the first two classes and the last two classes in Fig. 1 and 2 showed similar patterns. The systems are ordered from right to left along a gradient of increasing predator:prey biomass ratio. The resident systems showed higher predation level, in particular where lions make most of the carnivore biomass (Kruger and Serengeti resident). The highest herbivory levels were calculated in resident system largely dominated by megaherbivores (Hwange) or in the Serengeti migratory system.

Our estimates predict that some herbivore species should disappear quickly from overexploitation by carnivores. This is unrealistic and could be explained by a potential underestimation bias in herbivore census data (sometimes by up to 30%: Jachmann 2002). Additionally, our estimates of annual predation rates were constant and came from instantaneous estimates even though predation rates are likely to decrease as prey become scarcer. Nevertheless, the estimated levels of predation are large enough to suggest that predation strongly regulates populations of herbivore species below 150 kg, since for many species our estimates predict that virtually all newborn individuals could be consumed by predators. If predation were entirely compensatory, that is, predated animals would die anyhow, then predation would not act as a limiting process [START_REF] Owen-Smith | Predator-prey size relationships in an African large-mammal food web[END_REF]. This assertion, however, appears highly unlikely given the high predation levels estimated here.

Our results also suggest that the relative importance of top-down and bottom-up processes may vary greatly between systems, even though species composition was very similar, suggesting a crucial role of the relative abundance of the various body size classes. The Serengeti resident system and the Kruger system both showed greater levels of consumption of herbivores by carnivores than of plants by herbivores, whereas the reverse was true in Ngorongoro and Hwange. The main feature that distinguishes these two groups of ecosystems is not their overall abundance of predators, since Ngorongoro has the highest and Hwange the lowest abundance of predators, but their different predator:prey biomass ratios, with higher ratios associated with higher herbivore consumption by carnivores. Also, the largest carnivore, lion, makes up most of the predator biomass in the Serengeti and Kruger whereas the spotted hyaena Crocuta crocuta makes up most of the predator biomass in Hwange and Ngorongoro. Interestingly, our comparative data suggest the prevalence of size-nested predation (sensu [START_REF] Hopcraft | Herbivore, resources and risks: alternating regulation along primary environmental gradients in savannas[END_REF]: the prey base of small predators is nested within that of larger predators) within the large African predator guild. In this situation an increase in the relative abundance of the largest carnivore (lions) increases predation pressure on the larger herbivores without freeing smaller herbivores from predation.

In systems with resident herbivores, the total consumption of primary production was B40%, which is consistent with existing studies on other terrestrial ecosystems [START_REF] Halaj | Terrestrial trophic cascades: how much do they trickle? Á[END_REF]. The largest effect on primary production occurred in the system dominated by megaherbivores, which were virtually free of predators. A singular exception is the Serengeti where medium-sized grazers avoid predation by migrating [START_REF] Fryxell | Aggregation and migration by grazing ungulates in relation to resources and predators[END_REF] and consume almost all of the available primary production. Theoretical models predict that this is conducive to grazing optimization [START_REF] De Mazancourt | Grazing optimization and nutrient cycling: when do herbivores enhance plant production?[END_REF], and the Serengeti is one of the best studied examples of this phenomenon [START_REF] Mcnaughton | Ecology of a grazing ecosystem: the Serengeti[END_REF]. Resident herbivores, however, consume only a minor proportion of primary production, possibly being held in check by predator populations boosted by the seasonal influx of migratory prey.

The main functional differences between the ecosystems considered in our study were their different predator:prey biomass ratios and different body size distributions within and across trophic levels. The distribution of herbivore body sizes within an ecosystem depends on the abiotic template, particularly as small herbivores are strongly constrained by food quality [START_REF] Illius | Modelling the nutritional ecology of ungulate herbivores: evolution of body size and competitive interactions[END_REF], which may explain why megaherbivores dominate poor-soil ecosystems. The interplay between environmental gradients, herbivore body size, and predation pressure seems consistent with the conceptual framework proposed by [START_REF] Hopcraft | Herbivore, resources and risks: alternating regulation along primary environmental gradients in savannas[END_REF]. Here we provide the comparative analysis called for by these authors, and demonstrate the strength of a food web approach to the study of large vertebrate communities. Our approach supports the importance of size-structured predation on consumption fluxes across trophic levels in savanna ecosystems. Although predation rates were probably overestimated, they suggest top-down limitation of small-and medium-sized herbivores. Body size diversity may thus be a key component of ecosystem functioning, both within trophic levels (horizontal diversity) and between trophic levels (vertical diversity) [START_REF] Duffy | The functional role of biodiversity in ecosystems: incorporating trophic complexity[END_REF].

Our results also suggest that the massive decline of very large herbivores [START_REF] Surovell | Global archaeological evidence for proboscidean overkill[END_REF] as well as the persecution of large predators by humans [START_REF] Berger | Anthropogenic extinction of top carnivores and interspecific animal behaviour: implications of the rapid decoupling of a web involving wolves, bears, moose and ravens[END_REF] may have drastic effects on the dynamics and functioning of terrestrial ecosystems, similar to the simpler predatorÁprey system in North America [START_REF] Ripple | Wolves and the ecology of fear: can predation risk structure ecosystems?[END_REF]. Thus, it is likely that temperate terrestrial ecosystems that maintained a large diversity of predators before their extirpation, function today very differently than in the past [START_REF] Andersen | The future role of large carnivores in terrestrial trophic interactions: the northern temperate view[END_REF]. Future study of these ecosystems could benefit from the food web approach presented here, which integrates current knowledge and offers a powerful comparative framework. and Ngorongoro). Estimated population growth rates were first calculated for each species in each system, then averaged over lion dominated and hyaena dominated systems, and finally averaged per herbivore body mass class. The systems dominated by lions appear to have lower calculated growth rates, implying a greater predation pressure, from the smaller to even the very large herbivore species. a Includes Suids, which have much higher observed annual predation rate (80%).

Figure 1 .

 1 Figure 1. Reconstructed food webs for the four savanna ecosystems, distinguishing between the migratory and resident system in the Serengeti. The same five species of carnivores are present in all ecosystems, i.e. from left to right, the African wild dog Lycaon pictus (25 kg), the cheetah Acinonyx jubatus (45 kg), the hyaena Crocuta crocuta (60 kg), the leopard Panthera pardus (40 kg), and the lion Panthera leo (145 kg). In this figure leopard and hyaena are swapped along the body size gradient for clarity. The herbivores size classes are similar to those in Fig. 2: 10Á25, 40Á70, 90Á140, 160Á250, 300Á500, 700Á900, 1500 kg. Circle diameters represent the biomass (kg 100 km (2 ) of carnivore species and herbivore body mass classes. The values were transformed for graphical convenience (0.3â for carnivores and 0.5â for herbivores). Arrow widths are proportional to prey-predator consumption fluxes. For herbivores, units are in kg of dry matter d (1 km (2 , for carnivores units are in kg of fresh weight d (1 100 km (2 .

Figure 4 .

 4 Figure 4. Estimated population growth rates for the seven body mass classes of herbivores in ecosystems where lions comprised most of the carnivore biomass (Kruger and Serengeti) and those where hyaena comprised most of the carnivore biomass (Hwangeand Ngorongoro). Estimated population growth rates were first calculated for each species in each system, then averaged over lion dominated and hyaena dominated systems, and finally averaged per herbivore body mass class. The systems dominated by lions appear to have lower calculated growth rates, implying a greater predation pressure, from the smaller to even the very large herbivore species. a Includes Suids, which have much higher observed annual predation rate (80%).

Acknowledgements Á We thank the Director General of the Zimbabwe Parks and Wildlife Management Authority for providing the opportunity to carry out this research in Hwange National Park. This research was carried out within the HERD program (Hwange Environmental Research Development) funded by the French Ministry of Foreign Affairs, the French Embassy in Zimbabwe, the CIRAD and the CNRS, and within the BioFun and FEAR projects funded by the French National Research Agency (respectively ANR-05-BDIV-013-01 and ANR-08-BLAN-0022). ML acknowledges a Discovery grant of the Natural Sciences and Engineering Research Council of Canada. We thank N. Owen-Smith, P. Duncan, I. J. Gordon, A. R. E. Sinclair, J.-M. Gaillard and G. Hopcraft for their useful comments on earlier versions of the manuscript.


Victor Le Layec

Stéphane Hourdez

Oxygen consumption rates in deep-sea hydrothermal vent scale worms: effect of lifestyle, oxygen concentration, and temperature sensitivity

Oxygen consumption rates in deep-sea hydrothermal vent scale worms: effect of lifestyle, oxygen concentration, and temperature sensitivity

Victor Le Layec, Stéphane Hourdez

Introduction

Deep-sea hydrothermal vents are high-biomass ecosystems in comparatively barren surroundings [START_REF] Tunnicliffe | The Nature and Origin of the Modern Hydrothermal Vent Fauna[END_REF]. They rely on local primary production by chemo-autotrophic microorganisms that use the oxidation of reduced chemical compounds such as H2S, H2, CH4 and Fe 2+ contained in the hydrothermal fluid as an energy source [START_REF] Childress | The biology of hydrothermal vent animals: Physiology, biochemistry, and autotrophic symbioses[END_REF][START_REF] Van Dover | Microorganisms as food resources at deep-sea hydrothermal vents[END_REF].

Despite the seemingly attractive character of this environment, the conditions encountered there can be very challenging. In particular, the mixing of the hot hydrothermal fluid with the very cold surrounding deep-sea water is very chaotic and, as result, temperature and chemistry vary widely and rapidly [START_REF] Childress | The biology of hydrothermal vent animals: Physiology, biochemistry, and autotrophic symbioses[END_REF]. The pure hydrothermal fluid is devoid of oxygen, rich in CO2, low pH, and contains toxic compounds such as H2S, and heavy metals (Hg, Cd, Zn, …). As a result, organisms can be exposed to varying levels of hypoxia (low oxygen), hypercapnia (high CO2), and low pH. This is most likely one of the main drivers for the high rate of endemicity of the fauna found near deep-sea hydrothermal vents. Hypoxia is a major challenge, and its effect can be complicated by the positive correlation between temperature and hypoxia of the water surrounding the organisms [START_REF] Hourdez | Adaptations to hypoxia in hydrothermal-vent and cold-seep invertebrates[END_REF].

The few studies on metabolic needs suggest that deep-sea hydrothermal vent crabs and annelids [START_REF] Childress | Metabolic rates of animals from the hydrothermal vents and other deep-sea habitats[END_REF][START_REF] Hourdez | Adaptations to hypoxia in hydrothermal-vent and cold-seep invertebrates[END_REF]) have oxygen consumption rates similar to those of non-vent relatives. Other studies have focused on adaptations to chronic hypoxia in hydrothermal vent invertebrates, considering the morphology, physiology, and blood functional properties [START_REF] Hourdez | Adaptations to hypoxia in hydrothermal-vent and cold-seep invertebrates[END_REF]. Morphological studies have mostly focused on the gills of invertebrates, and showed that while vent-dwelling annelids possess increased gill surface areas, vent crustaceans usually do not [START_REF] Decelle | Morphological adaptations to chronic hypoxia in deep-sea decapod crustaceans from hydrothermal vents and cold seeps[END_REF][START_REF] Hourdez | Adaptations to hypoxia in hydrothermal-vent and cold-seep invertebrates[END_REF]. Similarly, gills from vent-dwelling annelids exhibit reduced diffusion distances that facilitate the diffusion of gases [START_REF] Hourdez | Adaptations to hypoxia in hydrothermal-vent and cold-seep invertebrates[END_REF] but vent-dwelling crustaceans do not display this adaptation [START_REF] Decelle | Morphological adaptations to chronic hypoxia in deep-sea decapod crustaceans from hydrothermal vents and cold seeps[END_REF]. Crabs and shrimp from hydrothermal vents however possess greater ventilatory capacity (increased scaphognathite surface area) compared to shallow-water temperate relatives [START_REF] Decelle | Morphological adaptations to chronic hypoxia in deep-sea decapod crustaceans from hydrothermal vents and cold seeps[END_REF]. Studies on Bythograea thermydron [START_REF] Gorodezky | Effects of sulfide exposure history and hemolymph thiosulfate on oxygen-consumption rates and regulation in the hydrothermal vent crab Bythograea thermydron[END_REF] and Segonzacia mesatlantica [START_REF] Hourdez | Cardiac response of the hydrothermal vent crab Segonzacia mesatlantica to variable temperature and oxygen levels[END_REF] have shown that these crabs can regulate their oxygen uptake to compensate for the decrease of environmental concentration down to very low levels compared to shallow-water relatives. This oxyregulation does not involve a modification of the heart rate but most likely relies on a modification of the ventilation rate [START_REF] Hourdez | Cardiac response of the hydrothermal vent crab Segonzacia mesatlantica to variable temperature and oxygen levels[END_REF].

Most hydrothermal vent organisms possess circulating respiratory pigments (hemocyanin and hemoglobin) in their body fluids [START_REF] Hourdez | Adaptations to hypoxia in hydrothermal-vent and cold-seep invertebrates[END_REF]. Interestingly, circulating respiratory pigments were even found in representatives of some taxonomic groups that were formerly known for lacking them (e.g. Polynoidae annelids; [START_REF] Hourdez | Adaptations to hypoxia in hydrothermal-vent and cold-seep invertebrates[END_REF]. The sheer presence of respiratory pigments could represent a form of oxygen storage for periods of anoxia. In addition, they can facilitate oxygen diffusion by binding this gas inside the body. This is further aided by the very high affinity for oxygen these molecules exhibit, leaving only very low partial pressures of free oxygen in the fluids [START_REF] Hourdez | Metazoan adaptation to deep-sea hydrothermal vents, in: Life in Extreme Environments[END_REF][START_REF] Hourdez | Adaptations to hypoxia in hydrothermal-vent and cold-seep invertebrates[END_REF].

The very dynamic nature of the vent environment, and in particular the rapid variations of temperature experienced by the deep-sea hydrothermal vent organisms has prompted a great deal of interest to understand the limits and tolerance mechanisms of response and adaptation to this parameter [START_REF] Hourdez | Metazoan adaptation to deep-sea hydrothermal vents, in: Life in Extreme Environments[END_REF]. In shallow-water marine environments, temperature follows a seasonal pattern, varying by about 2˚C throughout the year in Antarctic waters to nearly 20˚C in some temperate areas [START_REF] Sunday | Global analysis of thermal tolerance and latitude in ectotherms[END_REF]. In marine species, thermal tolerance breadth increases with latitude before decreasing near the poles [START_REF] Sunday | Global analysis of thermal tolerance and latitude in ectotherms[END_REF].

At deep-sea hydrothermal vents, species appear to be distributed in correlation with temperature, ranging from 2-3˚C (standard temperature of deep-sea water) to about 40˚C on the walls of chimneys [START_REF] Desbruyères | Données écologiques sur un nouveau site de hydrothermalisme actif de la ride du pacifique oriental[END_REF]. However, even at a fixed location, temperature can vary widely over short periods of time [START_REF] Bates | Deep-sea hydrothermal vent animals seek cool fluids in a highly variable thermal environment[END_REF][START_REF] Johnson | Chemical and biological interactions in the Rose Garden hydrothermal vent field, Galapagos spreading center[END_REF][START_REF] Johnson | In Situ Measurements of Chemical Distributions in a Deep-Sea Hydrothermal Vent Field[END_REF]. In this highly variable environment, metazoans seem to seek cooler conditions compared to shallow-water species in respect to their respective actual upper temperature tolerance [START_REF] Bates | Deep-sea hydrothermal vent animals seek cool fluids in a highly variable thermal environment[END_REF].

In situ observations usually provide a good picture of the thermal environment encountered by the different species but the very steep gradients of conditions (over centimeter scales) and the inherent difficulties of working in the deep sea represent a serious challenge to properly characterize the actual living conditions of these organisms. The species Alvinella pompejana, a hydrothermal vent chimney wall dweller, illustrates this well. An early observation showed a specimen curled around a temperature probe indicating 105˚C [START_REF] Chevaldonné | and some even hotter[END_REF], and in situ measurements suggested the worms were continuously exposed to temperature between 60 and 80˚C [START_REF] Cary | Worms bask in extreme temperatures[END_REF]. These values are greatly in excess of upper temperature limits measured for various biochemical processes in this species [START_REF] Chevaldonné | Thermotolerance and the "Pompeii worms[END_REF]. More recent work on live specimens in pressurized aquaria revealed that a 2 h exposure to 50-55˚C was lethal for the worms and that they were unlikely to withstand environmental temperatures greater than 50˚C for extended periods of time [START_REF] Ravaux | Thermal Limit for Metazoan Life in Question: In Vivo Heat Tolerance of the Pompeii Worm[END_REF], in better agreement with the aforementioned biochemical processes.

Annelids represent an important proportion of the endemic biodiversity at hydrothermal vents, and, among them, scale worms (Polynoidae) are the most diverse [START_REF] Tunnicliffe | The Nature and Origin of the Modern Hydrothermal Vent Fauna[END_REF]. These scale worms are found in all microhabitats where metazoans are present, from the coldest (and therefore most oxygenated) areas where the influence of the hydrothermal vent fluid is minimal, to the warmest possibly most hypoxic areas such as on chimney walls. Different species occupy different ranges of temperature, and of chemical conditions that are correlated to this temperature [START_REF] Desbruyères | Données écologiques sur un nouveau site de hydrothermalisme actif de la ride du pacifique oriental[END_REF][START_REF] Podowski | Biotic and abiotic factors affecting distributions of megafauna in diffuse flow on andesite and basalt along the Eastern Lau Spreading Center, Tonga[END_REF]. These species are usually free-living and can move to avoid adverse conditions, but species of the genus Branchipolynoe are commensal with mussels and therefore have limited mobility. This family is found in many other environments, from shallow-water to the deep-sea, from polar to tropical waters. It is very diverse, with about 900 species, and lends itself well to comparative work.

The present study aimed at determining the metabolic requirements and thermal tolerance of scale worms of the family Polynoidae from hydrothermal vents and compare them to shallowwater species of the same family. To that end, we worked with specimens placed in pressurized aquaria maintained at in situ pressure and measured oxygen consumption rates. We investigated the effect of life-style, environmental oxygen concentration and temperature on these rates to seek potential characteristics with an adaptive value in vent species.

Materials and methods

Three types of experiments were carried out: oxygen consumption rates, oxyregulation, and temperature sensitivity of oxygen consumption.

Sampling and abiotic parameters

Samples were collected from a variety of locations and habitats (Table 1). In total, 17 species were studied. Specimens were either collected near the Roscoff marine laboratory, or during different scientific expeditions on deep-sea hydrothermal vents: on the East Pacific Rise (cruises EPR2002, MESCAL 1, and MESCAL 2), on the Mid-Atlantic Ridge (TRANSECT cruise), and in the West Pacific (CHUBACARC cruise). Finally, a species was collected from an expedition at cold seeps in West Africa (WACS cruise). All experiments on deep-sea animals were performed onboard research vessels and those on shallow-water species in the lab. Details of the species, numbers of specimens for each type of experiments are provided in table 1. Typical temperatures encountered by each species are also indicated. Studies with in situ measurements of oxygen concentration values are very scarce and indicate that values typically range from 80±48 to 144±22 µmol.l -1 for different animal assemblages, to 175 µmol.l - 1 in ambient water [START_REF] Johnson | In Situ Measurements of Chemical Distributions in a Deep-Sea Hydrothermal Vent Field[END_REF][START_REF] Podowski | Biotic and abiotic factors affecting distributions of megafauna in diffuse flow on andesite and basalt along the Eastern Lau Spreading Center, Tonga[END_REF][START_REF] Podowski | Distribution of diffuse flow megafauna in two sites on the Eastern Lau Spreading Center, Tonga[END_REF]. No data are available for cold-seep assemblages and for hydrothermal-vent chimney walls. Shallow-water species are usually exposed to air-saturated water which concentration depends on temperature (200-260 µmol.l -1 ). All experiments used natural seawater (36-38 ppt) that was filtered on 1 µm (cartridge filters).

Oxyregulation

In a first set of experiments, we aimed to study the capacity of different species to compensate a decrease of environmental oxygen concentrations to meet their metabolic demand (oxyregulation). Each experiment was performed on a single individual in a flow-through system at in situ pressure (provided by High Pressure Liquid Chromatography pumps, see [START_REF] Hourdez | Cardiac response of the hydrothermal vent crab Segonzacia mesatlantica to variable temperature and oxygen levels[END_REF] and at a temperature of 10˚C. Once placed in the flow-through vessels, animals were allowed to recover for 10-12 hours before experimentation started. Oxygen concentration was measured with an optode (NeoFox, Ocean Optics) placed in the outflow of the system (i.e. after the pressure relief valve), either after flowing through the aquarium containing the specimen or after flowing through a tubing that bypassed the aquarium [START_REF] Hourdez | Cardiac response of the hydrothermal vent crab Segonzacia mesatlantica to variable temperature and oxygen levels[END_REF].

Oxygen consumption was calculated based on the water flow rate of the HPLC pump, and the difference of oxygen concentration between the aquarium and bypass paths. Runs without animals were used as controls to remove oxygen consumption due to bacteria. Oxygen concentration in the seawater feeding the aquarium was modified by bubbling either air (to reach air saturation) or nitrogen (to decrease the oxygen concentration) in the water. After outflow oxygen concentration reached a plateau, oxygen consumption was measured as described above. Oxygen concentration was ramped up or down in succession (i.e. not constantly going down) to mimic environmental concentration changes and avoid acclimation.. Overall, these experiments lasted for about 36 hours for each individual and were performed for 7 species and 9 individuals in total (3 specimens for B. aff. seepensis; see Table 1). These numbers include two shallow-water temperate species, one cold-seep species, and four deepsea hydrothermal vent species. No deep-sea non-vent species could be used for this type of experiment.

Oxygen consumption rates

In another set of experiments, simple oxygen consumption experiments were carried out.

For deep-sea species, two types of experiments were carried out. In the first type, hereafter referred to as 'closed vessel' the specimens were individually placed in a gas-tight Hamilton syringe that was in turn placed into a large pressure vessel. In the second type of experiments, a specimen was individually placed into a flow-through vessel as described above. For shallowwater species, the oxygen consumption was measured in the flow-through system only, allowing us to keep the environmental oxygen concentration provided constant. Temperature of incubation was controlled by placing the pressure vessel into a water-bath at the desired temperature (± 0.2˚C). All reported consumption rates were measured at 10˚C. The water used in the incubations and flow-through was bubbled with air contained typically 200-260 µmol.l - 1 of oxygen, depending on room temperature.

For the closed vessels, after 1.5 to 2.5 hours, a water sample was carefully withdrawn from the syringe containing the specimen and the gas contents of the water was measured with a gas chromatograph (GC) [START_REF] Arp | Sulfide binding by the blood of the hydrothermal vent tube worm Riftia pachyptila[END_REF]. Oxygen consumption was calculated based on the volume of water in the syringe, oxygen concentration difference, and duration of the incubation. Syringes that contained no animals were used as control for bacterial consumption.

For flow-through vessels, animals were allowed to recover for 10-12 hours before experimentation started. Oxygen consumption was calculated as described above for the oxyregulation experiments. Runs without animals were used as controls to remove oxygen consumption due to bacteria. Because the animals are able to move inside the pressure vessel and go between periods of acitivty and rest, oxygen consumption was measured every 2 hours, 3 or 4 times and the average consumption is reported.

Overall, each individual oxygen consumption experiment lasted between 3 hours for closed vessel experiments and 16-24 hours for flow-through experiments. A total of 99 specimens representing 17 species were used in these experiments. Details of specimens and species used for each type of oxygen consumption experiment are provided in Table 1.

The demonstration of the capacity to oxyregulate for hydrothermal vent species (see Results) allows us to directly compare measurements made in flow-through and closed chambers for these species.

Temperature sensitivity

In the last set of experiments, we measured oxygen consumption rates as temperature was regularly increased in the flow-through vessel (6 ˚C/h). Start temperature was typically 6˚C (close to deep-sea temperature at these depths) and the experiment was ended when the specimen displayed signs of death (ventriflexion and lack of movement). Most experiments ended at about 37˚C (about 6 hours total). For shallow-water species, start temperature was about 10˚C to not induce cold stress and was ended when the animals displayed signs of death.

The experiments ended at about 30˚C, for a total duration of about 2.5 hours. The setup was the same as described above, the source seawater was continuously bubbled with air to maintain air saturation (ca. 260 µmol.l -1 oxygen in inlet), and the oxygen consumption was measured in the outlet after the pressure-relief valve. To reflect the highly variable conditions encountered at hydrothermal vents, no acclimation period was given to the specimens. This experiment was performed on a total of 9 specimens (see tables 1 and 2), including one shallowwater species (one specimen), one deep-sea (one specimen) and 4 hydrothermal vent species (one or two specimens each). This type of experiment was not performed on the cold-seep species. Because the specimens went through periods of activity and rest, it was not possible to determine an Arrhenius Break Point (ABP).

Experimental specimen preservation and weighing

All animals were preserved after in 85% ethanol after the experiments and their wet weight was measured on an analytical balance (Mettler Toledo, precision 0.001 g) after returning to the lab.

Statistical analyses

For oxyregulation experiments, the curve described in [START_REF] Hourdez | Cardiac response of the hydrothermal vent crab Segonzacia mesatlantica to variable temperature and oxygen levels[END_REF] was used to fit to the datapoints and determine the oxygen concentration below which oxyregulation was no longer possible (critical oxygen concentration).

The relationship between oxygen consumption rates and wet weight was linearized using a log/log transform. Because some species and groups are represented by few individuals, a linear regression was used on the whole dataset and deviations from this regression were used for comparison. The residuals to this relationship were calculated and compared to see whether species from different habitats had different oxygen requirements.

Results

Capacity to oxyregulate

We measured oxygen consumption rates and the effect of oxygen concentration on this consumption at 10˚C for four different species from deep-sea hydrothermal vents, a cold-seep species (at 4˚C), and two shallow-water temperate species (Figure 1). For the coastal species Pettibonesia furcosetosa and Harmothoe extenuata (Figure 1A), over the range 200 µmol.l -1 to 50 µmol.l -1 , the consumption decreases with the concentration of oxygen in the environment.

In contrast, over the same range, all hydrothermal vent species and the cold-seep species (Figure 1B) the oxygen consumption remains relatively constant. Below this lower concentration, there is a sharp drop at about an oxygen concentration of 30 µmol.l -1 in the environment.

The capacity to extract the same amount of oxygen from the environment regardless of its concentration (oxyregulation) has been observed for all hydrothermal vent species, including Lepidonotopodium fimbriatum, which does not possess gills (Figure 1B). For Branchinotogluma segonzaci (Figure 1B), we did not reach the limit below which the animal was not able to maintain its oxygen consumption (lowest oxygen concentration of 30 µmol.l -1 in the aquarium). The value below which the other species are no longer capable of oxyregulation and oxygen consumption reaches zero is about 25 µmol.l -1 for Lepidonotopodium fimbriatum (Figure 1B) and Branchipolynoe aff. seepensis (Figure 1B), and probably lower for Thermopolynoe branchiata (Figure 1B), although there are too few data points below that value for confidence.

Effect of size, life-style and habitat of origin

We measured oxygen consumption rates at 10˚C for 99 specimens representing 2 species from deep-sea non-vent areas, 11 from deep-sea hydrothermal vents, and three shallow-water temperate species (Figure 2). For all specimens pulled together, there is a correlation between the consumption rate and the size of the animal (Figure 2A), and the specific oxygen consumption rate (per gram wet weight) decreases as the wet weight of the animal increases (r 2 =0.2481, p<0.001, slope -0.353).

To detect whether life style or the environment had an effect of the oxygen consumption rates, we calculated the residuals to the correlation established for the whole dataset (Figure 2B). All deep-sea hydrothermal vent (free-living and commensal) and non-hydrothermal vent abyssal species have a similar oxygen consumption (p-value < 0.05). Although difficult to directly compare because the consumption of the shallow-water species depends on oxygen concentration and the limited availability of coastal replicates for this comparison, the coastal specimens do not have a significantly higher consumption rate than the deep-sea ones (Oneway ANOVA p < 0.0001). The cold-seep species was studied at 4˚C and its consumption rates cannot be directly compared to all the other species (experiments performed at 10˚C).

Effect of temperature on oxygen consumption rates

During the experiments to study the effect of temperature, the animals went through phases of activity and rest, which manifested as rises and falls in oxygen consumption (Figure 3).

Overall, throughout the experiment, their activity increased with temperature (pers. obs.). At higher temperature values, we observed spasms and ventriflexion which are characteristics of imminent death of the animals. None of the experimental animals survived temperatures greater than 38°C. For the shallow-water species, death occurred at much lower temperature than vent species (ca. 27˚C; Figure 3A). For all species, the oxygen consumption rate increases with temperature.

Overall, the slopes of the linear part ranged from -1.17 for the shallow-water species Gattyana cirrhosa to -3.49 for one specimen of the hydrothermal vent chimney species Branchinotogluma segonzaci (Supplementary figure 1 andTable 2). For three of the six species tested, two specimens were tested and showed some variability in response to increasing temperature. For two of the species, the slope only varies by 0.3-0.6 but for B. segonzaci, the two specimens yielded two very different slopes (-1.56 and -3.49; supplementary figure 1B andC). For all hydrothermal vent species, the slope was greater (i.e. the oxygen consumption rates are more sensitive to temperature variation) than the shallow-water temperate species Gattyana cirrhosa and the deep-sea species Thermiphione sp.. At higher temperature, oxygen consumption reaches an inflexion point that could be interpreted as the Arrhenius break point (ABP), although the slope rupture is not sharp and an accurate determination is not possible (Figure 3).

Discussion

Oxyregulation in hydrothermal vent species

Animals can be classified as either oxyregulators or oxyconformers according to their respiratory response to hypoxia [START_REF] Prosser | PHYSIOLOGICAL VARIATION IN ANIMALS[END_REF]. Oxyregulators have the capacity to regulate their oxygen uptake to compensate variations of the environmental concentration of this gas, while oxygen consumption is proportional to environmental oxygen concentration in oxyconformers. While anaerobic energy production starts at high oxygen partial pressures for oxyconformers, oxyregulators do not rely on this metabolism until a partial pressure under which oxygen consumption decreases sharply and the organism increasingly relies on anaerobic metabolism. Our data on Polynoidae show that the littoral species Pettibonesia furcosetosa and Harmothoe extenuata fall into the oxyconformer category, while all hydrothermal vent species tested for this capacity are oxyregulators. Sthenelais boa, a species of the closely-related scale worm family Sigalionidae, also falls into the oxyconformer category [START_REF] Cosgrove | The effect of environmental oxygen concentration on the rate of oxygen consumption of the polychaete Sthenelais boa (Johnston)[END_REF]. The fact that oxyregulation was so far only encountered in hydrothermal vent species could indicate that this is a derived character in these species. This interpretation is consistent with our current understanding of scale worm phylogeny [START_REF] Norlinder | Phylogeny of scale-worms (Aphroditiformia, Annelida), assessed from 18SrRNA, 28SrRNA, 16SrRNA, mitochondrial cytochrome c oxidase subunit I (COI), and morphology[END_REF], in which the vent species form a monophyletic group and the two shallow-water species of Polynoidae and the Sigalionidae are basal. However, additional non-vent Polynoidae species and members of closely related families need to be studied to reliably determine the ancestral state of the response to varying environmental oxygen concentrations.

The capacity to oxyregulate relies on compensatory mechanisms that can involve ventilation, diffusion, or circulatory responses (see [START_REF] Hourdez | Adaptations to hypoxia in hydrothermal-vent and cold-seep invertebrates[END_REF] for a review on adaptation to chronic hypoxia). In general, in scale worms, including Polynoidae, water circulation is produced by ciliary movements at the surface of the body. In the shallow-water species Halosydna brevisetosa, the elytra form a roof that allows a directional and effective flow of water from the front to the posterior end of the animal [START_REF] Lwebuga-Mukasa | The role of elytra in the movement of water over the surface of Halosydna brevisetosa (Polychaeta: Polynoidae)[END_REF]. Scale worms usually lack gills but some hydrothermal vent species possess segmental coelomic gills [START_REF] Hourdez | Functional anatomy of the respiratory system of Branchipolynoe species (Polychaeta, Polynoidae), commensal with Bathymodiolus species (Bivalvia, Mytilidae) from deep-sea hydrothermal vents[END_REF]. These gills increase the gas exchange surface area and offer reduced diffusion distances compared to the typical body-wall through which gas exchange occurs in other species of scale worms. Our work shows that regardless of the presence of gills, all tested hydrothermal vent species are capable of oxyregulation, and that this character does not seem to affect the critical partial pressure of oxygen below which the animals can no longer compensate. In the genus Branchipolynoe, elytra are usually reduced in size but they still cover the gills (that are attached to the parapodia) and likely participate in the efficient water flow over the gills (that bear numerous cilia; [START_REF] Hourdez | Functional anatomy of the respiratory system of Branchipolynoe species (Polychaeta, Polynoidae), commensal with Bathymodiolus species (Bivalvia, Mytilidae) from deep-sea hydrothermal vents[END_REF], where most of the oxygen exchange occurs. This genus is also commensal in the mantle cavity of bathymodiolin mussels, and can benefit from the water flow produced by their host.

A common character of hydrothermal vent species compared to shallow-water species is the presence of hemoglobin in large amounts in their coelomic fluid (Hourdez et al., 1999b(Hourdez et al., , 1999a;;[START_REF] Projecto-Garcia | Evolution of single-domain globins in hydrothermal vent scale-worms[END_REF]. In contrast, shallow-water species all lack a circulating respiratory pigment and only exhibit small amounts of globin in their nervous system (Hourdez et al., 1999a;[START_REF] Weber | Respiration[END_REF]. The vascular system is poorly developed in all scale worms and oxygen diffuses through the epidermis to reach the coelomic fluid that bathes all the internal organs. The coelomic epithelium is ciliated and therefore the fluid circulates. It is unknown whether this circulation rate can be modified as a possible response to low oxygen values.

Hemoglobins in the species studied so far exhibit a high affinity for oxygen that likely facilitates oxygen diffusion into the body (Hourdez et al., 1999b(Hourdez et al., , 1999a;;[START_REF] Projecto-Garcia | Evolution of single-domain globins in hydrothermal vent scale-worms[END_REF]. The presence of high-affinity hemoglobins however is not always associated with the capacity to oxyregulate. In the giant tubeworm Riftia pachyptila (Annelida, Siboglinidae) for example, oxygen consumption is directly proportional to oxygen partial pressure (=oxyconformers; [START_REF] Girguis | Metabolite uptake, stoichiometry and chemoautotrophic function of the hydrothermal vent tubeworm Riftia pachyptila: responses to environmental variations in substrate concentrations and temperature[END_REF]. Conversely, some invertebrates devoid of respiratory pigments such as mussels can oxyregulate if the temperature is not too high [START_REF] Hourdez | Characterization and functional properties of the extracellular coelomic hemoglobins from the deep-sea, hydrothermal vent scaleworm Branchipolynoe symmytilida[END_REF]. [START_REF] May | THE EFFECTS OF OXYGEN CONCENTRATION AND ANOXIA ON RESPIRATION OF ABARENICOLA PACIFICA AND LUMBRINERIS ZONATA (POLYCHAETA)[END_REF] showed that two species of annelids (Abarenicola pacifica (Arenicolidae) and Lumbrineris zonata (Lumbrineridae)) exhibited an oxyregulatory capacity in summer but not in springtime. In our case, we did not test this capacity in summertime and it is possible that shallow-water species could exhibit an oxyregulatory capacity in summer. If this capacity exists, the absence of high-affinity respiratory pigments would likely limit the critical partial pressure for these species. In the hydrothermal bythograeid crabs Bythograea thermydron and Segonzacia mesatlantica, the critical partial pressure can be linked to the high affinity of the hemocyanin (Gorodevsky and [START_REF] Gorodezky | Effects of sulfide exposure history and hemolymph thiosulfate on oxygen-consumption rates and regulation in the hydrothermal vent crab Bythograea thermydron[END_REF][START_REF] Hourdez | Cardiac response of the hydrothermal vent crab Segonzacia mesatlantica to variable temperature and oxygen levels[END_REF]. Shallow-water species of crabs can also oxyregulate but the critical partial pressure is much higher, reflecting the much lower affinity of their hemocyanin for oxygen (e.g. critical oxygen concentration 100-130 µmol.l -1 in Carcinus maenas; [START_REF] Taylor | The respiratory responses of Carcinus maenas to declining oxygen tention[END_REF].

Effect of natural habitat, lifestyle, and size

The capacity to oxyregulate allows us to directly compare oxygen consumption rates obtained from flow-through (shallow-water, some East Pacific Rise measurements and West Pacific species) and closed-vessel experiments (remaining East Pacific Rise measurements).

Overall, once the weight of specimens and temperature are considered, the oxygen consumption rates measured here are well in the typical range reported for other annelids [START_REF] Weber | Respiration[END_REF][START_REF] Childress | Metabolic rates of animals from the hydrothermal vents and other deep-sea habitats[END_REF]. The specific consumption rates are slightly higher for shallow-water species at high environmental oxygen concentration but drops below that of deep-sea species at values lower than 150 µmol.l -1 oxygen. The lack of oxyregulation capacity for shallow-water species therefore makes it difficult to compare oxygen consumption rates.

All consumption rates were measured using air-saturated water (ca. 260 µmol.l -1 at the temperature at which the experimental system was maintained). It is not clear whether deepsea, non-vent species are capable to oxyregulate but their specific consumption rates seem to be slightly lower than in shallow-water species (although the difference is not significant).

Oxygen consumption increases with activity level, including in tube-dwelling groups [START_REF] Weber | Respiration[END_REF]. Although Branchipolynoe, a genus that is commensal with bathymodiolin mussels, could be expected to have a lower activity level than free-living species, there is no significant difference of specific oxygen consumption rate between these two lifestyles at 10˚C. Besides the difference in oxyregulation, all species studied here consume very similar amounts of oxygen. The absence of such difference between deep-sea and shallow-water relatives was already reported in other taxonomic groups by [START_REF] Childress | Metabolic rates of animals from the hydrothermal vents and other deep-sea habitats[END_REF]: crustaceans, bivalves, and an array of polychaeta. These authors concluded that the metabolic rates of benthic deep-sea animals are not related to the food availability. Hydrothermal vent communities indeed benefit from large quantities of locally produced biomass (by autotrophic bacteria) compared to the very small amounts of photosynthetic biomass falling from surface.

This contrasts with pelagic groups with image-forming eyes that exhibit a decrease of metabolic rates with increasing depth (related to changes in locomotor capacities), although evolutionary history also has a strong importance [START_REF] Seibel | On the depth and scale of metabolic rate variation: scaling of oxygen consumption rates and enzymatic activity in the Class Cephalopoda (Mollusca)[END_REF].

As typically observed in other animals, while the oxygen consumption rate increases with body size, the specific oxygen consumption rate (i.e. consumption per gram body weight) decreases with body size. The higher oxygen consumption rate could reflect a higher investment in growth in smaller individuals. For our dataset, the relationship between size and specific oxygen consumption rates follows an allometric relationship with a coefficient of 0.35, close to the value reported in the classical studies of the 1960's for annelids (e.g. 0.33 in [START_REF] Banse | Oxygen consumption by the seabed. III. On the role of the macrofauna at three stations[END_REF]. This value indicates that the metabolic rate is proportional to the surface area of the animals rather than their body weight [START_REF] Weber | Respiration[END_REF].

Effect of temperature

Temperature constrains every biological process, in particular metabolic processes [START_REF] Hochachka | Biochemical adaptation[END_REF]. Accordingly, oxygen consumption rates increased with temperature for all species tested. Because of the succession of rest and active periods throughout the experiment, the determination of an Arrhenius Break Point was difficult. However, shallow-water species died in slightly lower temperature than deep-sea species (27˚C vs. 30-38˚C, respectively).

More than absolute values, temperature variability and the organisms' response to this variability may be limiting their distribution around hydrothermal vents. In earlier experiments, authors have shown that eurythermal species typically exhibit less sensitivity to temperature variation than species that naturally experience a more limited range of temperature (see [START_REF] Mangum | Temperature adaptation[END_REF]. These studies, however, were performed on species that experience either very stable temperatures (general deep-sea) or seasonal changes of temperatures, and specimens were given 9-21 days of acclimation at the desired temperature. Deep-sea hydrothermal vent species are exposed to temperature changes that can reach 10 degrees over a few minutes. In our study, specimens were not acclimated to the desired temperature to better mimic the hydrothermal vent environment. Contrary to our expectations, while inhabiting a highly variable environment, the oxygen consumption of the deep-sea hydrothermal vent species exhibited a greater sensitivity to temperature variation (greater Arrhenius slope values).

A single shallow-water temperate species was studied for this response, and additional species should to be investigated to confirm this observation. The marked variability observed within a species could be evidence for acclimation, the replicates coming from distinct collections. This would also need to be confirmed through further measurements on specimens from a single collection point, and from distinct sites. All hydrothermal vent species were very active in the aquaria when the temperature increased, while the shallow-water species, although more active, did not show such a higher degree of activity (pers. obs.). This difference of behaviour could be a reflexion of the early onset of avoidance behaviour of hydrothermal vent species which attempt to remain at temperatures well below their lethal threshold compared to temperate species [START_REF] Bates | Deep-sea hydrothermal vent animals seek cool fluids in a highly variable thermal environment[END_REF]. These authors indicate that avoidance of hot conditions is a primary defence strategy used by ectotherms from the tropics and deserts but not specifically displayed by temperate marine fauna. This latter fauna is more likely to experience tidal or seasonal thermal variations but not the rapid and chaotic spikes of temperature that characterize hydrothermal vents. As a consequence, for instance, most vent molluscs (provannids, limpets or bivalves) are usually located at a distance from vent flows, with temperature variability oscillating between 5 and 15°C, and other species represent true stenothermal abyssal taxa, with temperatures that do not exceed 4°C [START_REF] Bates | Role of thermal conditions in habitat selection by hydrothermal vent gastropods[END_REF][START_REF] Bates | Deep-sea hydrothermal vent animals seek cool fluids in a highly variable thermal environment[END_REF].

The oxygen and capacity-limited thermal tolerance (OCLTT) concept provides an explicative framework to study and understand the thermal range of aquatic animal species [START_REF] Pörtner | Oxygen-and capacity-limited thermal tolerance: bridging ecology and physiology[END_REF]. In this concept, oxygen limitation takes a central role in the thermal tolerance of species.

The capacity to oxyregulate for species from hydrothermal vents likely also provides them a better thermal tolerance or a wider thermal range than for shallow-water species.

Conclusions

In conclusion, for the species investigated in this study, only hydrothermal vent species can oxyregulate down to oxygen concentrations as low as 30 µmol.l -1 , while shallow-water species are oxyconformers. Oxygen consumption rates are not different comparing contrasted lifestyles (free-living vs. commensal, shallow-water vs. deep-sea vs. hydrothermal vent). Surprisingly, although hydrothermal vent species are exposed to highly variable temperatures (ca. 10˚C over a few minutes), their oxygen consumption rates do not exhibit a reduced sensitivity to temperature variation. On the contrary, oxygen consumption increases more strongly with temperature in vent species. This could provide higher energy for the animals to escape from conditions too close to lethal temperatures.

Table 1 :

 1 Specimens used in this study. Species listed in alphabetical order, expedition, habitat, depth, and range of typical temperatures encountered. A range of wet weights is provided for each species. CVR= Closed Vessel Respirometry; FTR= Flow-Through Respirometry; Temp= effect of Temperature; Oxy= Oxyregulation.

	Species	Sampling expedition	Habitat type	Site name	Coordinates	Depth	Temp	Specimens	Wet
						(m)	range (˚C)	studied	weight (g)
	Branchinotogluma grasslei	EPR2002	Hydrothermal vent 9˚N	9˚49.80'N 104˚17.40'W 2700	10-25	2 CVR	0.347*
	Branchinotogluma hessleri	EPR2002	Hydrothermal vent 9˚N	9˚49.80'N 104˚17.40'W 2700	10-25	1 CVR	0.037
	Branchinotogluma sp. nov.	CHUBACARC	Hydrothermal vent Mangatolo	15˚57.24'S 174˚42.66'W 1330	3-5	1 FTR	0.385
	Branchinotogluma segonzaci	CHUBACARC	Hydrothermal vent Solwara 8	3˚43.50'S 151˚40.20'E	1500	20-30	2 FTR,	0.310-
								2 Temp,	2.761
								1 Oxy
	Branchinotogluma trifurcus	CHUBACARC	Hydrothermal vent Tu'i Malila	21˚59.26'S 176˚34.07'W	1850	10-20	4 FTR,	0.132-
				Tow Cam	20˚19.01'S 176˚08.20'W	2700		1 Temp	0.205
	Branchipolynoe tjiasmantoi	CHUBACARC	Hydrothermal vent Kulo Lasi	14˚55.20'S 177˚15.00'W	1480	6-8	3 FTR,	0.463-
				Tu'i Malila	21˚59.26'S 176˚34.07'W	1850		2 Temp	1.786
				ABE	20˚45.71'S 176˚11.46'W	2200
	Branchipolynoe aff. seepensis WACS	Cold seeps	REGAB	5˚46.89'S 9˚44.66'E	3200	4	3 FTR,	1.288-
								3 Oxy**	1.552
	Branchipolynoe symmytilida	MESCAL, EPR2002 Hydrothermal vent 9˚N	9˚49.80'N 104˚17.40'W 2700	6-8	42 CVR,	0.008-
								1 FTR,	4.650
								1 Oxy
	Eulagiscinae gen. sp.	CHUBACARC	Deep sea, non-vent Fatu Kapa	3˚43.50'S 151˚40.20'E	1500	4	1 FTR	0.346
	Gattyana cirrhosa	Roscoff Marine lab	Coastal temperate Penpoul	48˚40.77'N 3˚56.89'W	0-10	12-20	1 FTR	0.807
	Harmothoe extenuata	Roscoff Marine lab	Coastal temperate Ile Verte	48˚43.76'N 3˚59.24'W	0-10	12-20	2 FTR	0.134-
								1 Oxy	0.361
	Lepidonotopodium fimbriatum MESCAL	Hydrothermal vent 9˚N	9˚49.80'N 104˚17.40'W 2700	25-30	2 FTR,	0.553-
								1 Oxy	0.624
	Lepidonotopodium williamsae MESCAL,	Hydrothermal vent 9˚N	9˚49.80'N 104˚17.40'W 2700	12-15	4 CVR	0.311-
		MESCAL2, EPR2002							1.163
	Levensteiniella raisae	CHUBACARC	Hydrothermal vent Solwara 8	3˚43.5'S 151˚40.2'E	1520	7-10	7 FTR	0.102-
				Tu'i Malila	21˚59.26'S 176˚34.07'W	1800			5.126

Acknowledgements

We are grateful to the chief scientists, ship crews, and deep submersible pilots and technicians for the access to the specimens used in this study. We would like to thank Jim Childress for not only allowing us to use his equipment on the EPR2002 cruise but also kindly offering his critical reading of this manuscript. Many thanks to Cécile Cathalot for lending us an optode in replacement of a defective one during the CHUBACARC cruise. Thank you to Alexis Lecoeur for his help running experiments during the MESCAL cruise. This research was supported by the Région Bretagne HYPOXEVO program, the CERBERUS research program (ANR-17-CE02-0003), the Fonds Flotte Océanographique, and the Flotte Océanographique Française.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. for all species except the cold-seep species (4˚C) in the flow-through system. Oxygen concentration was modified in the inlet water by bubbling either pure nitrogen or air.

Experiments lasted up to 36 hours. For the X-axis, the inverse of temperature (in Kelvin) was multiplied by 1000 and the axis was reversed to keep low temperature values to the left for ease of reading. Corresponding axis with temperatures in ˚C is provided on top of each graph. A. Gattyana cirrhosa (shallow-water temperate), B. Branchipolynoe tjiasmantoi (hydrothermal vent mussel commensal), and C.

Thermopolynoe branchiata (hydrothermal vent, free-living). See supplementary figure 1 for the remaining plots. Oxygen consumption rate (µmol.h -1

)

Oxygen concentration (µmol.l -1 ) Supplementary figure 1