Kevin Sanchez-Thirion

Michaël Danger

Alexandre Bec

Elise Billoir

Sophie Labaude

Thierry Rigaud

Jean-Nicolas Beisel

Vincent Felten
email: vincent.felten@univ-lorraine.fr

Michael Danger

High food quality increases infection of Gammarus pulex (Crustacea: Amphipoda) by the acanthocephalan

Keywords: Parasite, food quality, ecological stoichiometry, experimental infestation, 26 survival, growth, behavior 27

come

Introduction

Parasitism is certainly one of the most important but largely neglected processes in ecosystems [START_REF] Lafferty | Parasites in food webs: The ultimate missing links[END_REF]. Parasites, ranging from bacteria and protozoans to metazoan species such as crustaceans or nematodes, use their hosts as providers of resources and habitats. Recent studies suggested that parasites may represent at least half of species on Earth, and could be involved in 75% of the trophic links in food webs [START_REF] Dobson | Homage to Linnaeus: How many parasites? How many hosts[END_REF].

To date, numerous studies have investigated the often complex life cycles of parasites and the physiological mechanisms involved in hosts manipulation. Far less is known on the impacts of environmental stressors on parasites success and, in turn, on the impacts of parasites on food webs and ecosystem processes [START_REF] Moore | Parasites and host behaviour[END_REF]Labaude et al., 2015a).

In all ecosystems, parasites' hosts are simultaneously exposed to several environmental stressors, either from natural or anthropic origins, and data on the interactive impacts of these stressors and parasites are clearly lacking. Among the different stressors that hosts might be exposed to, resources availability is certainly one of the most common [START_REF] Power | Top-down and bottom-up forces in food webs: do plants have primacy[END_REF]. Several studies have investigated the impact of resources quantity on host-parasite interactions [START_REF] Wright | Altered parasite assemblages in raccoons in response to manipulated resource availability[END_REF][START_REF] Civitello | Resources, key traits, and the size of fungal epidemics in Daphnia populations[END_REF]. Some authors also explained current increases in parasitic and infectious diseases by increased nutrient loading in the environment [START_REF] Johnson | Linking environmental nutrient enrichment and disease emergence in humans and wildlife[END_REF]). Yet, in numerous ecosystems, resource quality has been shown as far much important than resource quantity for understanding organisms' physiology, food web structure, and ecosystem functioning (Elser et al., 1998;[START_REF] Frost | Growth responses of littoral mayflies to the phosphorus content of their food[END_REF][START_REF] Marcarelli | Quantity and quality: unifying food web and ecosystem perspectives on the role of resource subsidies in freshwaters[END_REF]. Although investigating resources quality parameters instead of quantifying resources availability might be essential for understanding host-parasite interactions, data on this topic remain extremely scarce.

Several parameters are generally considered for describing resources quality. Resources elemental content, generally expressed as resources carbon (C): nitrogen (N): phosphorus (P) ratios, have regularly been shown to control consumers' life history traits, especially for herbivorous [START_REF] Elser | Nutrient limitation reduces food quality for zooplankton: Daphnia response to seston phosphorus enrichment[END_REF][START_REF] Schade | Stoichiometric tracking of soil nutrients by a desert insect herbivore[END_REF] or detritivorous [START_REF] Danger | Phosphorus content in detritus controls life-history traits of a detritivore[END_REF][START_REF] González | Detrital nutrient content determines growth rate and elemental composition of bromeliad-dwelling insects[END_REF][START_REF] Fuller | Growth and stoichiometry of a common aquatic detritivore respond to changes in resource stoichiometry[END_REF] species. This approach, called Ecological Stoichiometry [START_REF] Sterner | Ecological Stoichiometry: the biology of elements from molecules to the biosphere[END_REF], specifically investigates the consequences of elemental imbalances between consumers' requirements and elements available in resources.

Since parasites rely on their hosts for resource acquisition, optimal elemental composition of resources for an infected host might fulfill both parasites requirements but also host minimal requirements for staying alive [START_REF] Aalto | A three-way perspective of stoichiometric changes on host-parasite interactions[END_REF][START_REF] Bernot | Ecological stoichiometry for parasitologists[END_REF]. In a recent opinion paper, [START_REF] Bernot | Ecological stoichiometry for parasitologists[END_REF] highlighted the interest of ecological stoichiometry as a pertinent framework for studying feedbacks of parasites on the environment as well as the effect of the environment on parasites and diseases. Indeed, elemental requirements of infected hosts might thus largely differ from those of non-parasitized ones (Frost et al., 2008a), and, in turn, greatly alter the response of infected organisms to resources elemental quality (Frost et al., 2008b;[START_REF] Aalto | Food stoichiometry affects the outcome of Daphnia-parasite interaction[END_REF]. For example, Frost et al. (2008b) showed that Daphnia fed with phosphorus rich resources had higher infection rates and parasite loads than those fed with phosphorus-poor resources, this observation being explained by a phosphorus-limitation of parasites' growth rates. Beyond a quantity aspect, an indicator of resources quality is their lipid profiles, especially resource contents in polyunsaturated fatty acids (Arts et al., 2009). Indeed, some of these fatty acids are considered as essential for consumers, since they cannot be synthesized or at least not in sufficient amounts to fulfill the requirements of consumers (Arts et al., 2009). Availability of long chain PUFA in resources have been regularly reported as controlling consumers' growth and/or reproduction, especially in aquatic ecosystems where they can mainly be found in certain algal taxa (e.g. diatoms) or some protozoans (Müller-Navarra et al., 2004;[START_REF] Bec | Trophic upgrading of autotrophic picoplankton by the heterotrophic nanoflagellate Paraphysomonas sp[END_REF]. Moreover some long chain PUFA such arachidonic acid and eicosapentaenoic acid serve as biosynthetic precursor of eicosanoids known to be signalling molecules in many physiological processes, mostly related to reproduction and immunity (Stanley-Samuelason 2006). This lead [START_REF] Schlotz | Dietary supply with polyunsaturated fatty acids and resulting maternal effects influence host-parasite interactions[END_REF] to hypothesize that a high dietary supply with PUFA may result in more pronounced immune response to the parasite resulting in increased resistance to infection.

Among the numerous invertebrate species inhabiting freshwater ecosystems, the crustacean Gammarus pulex (Crustacea: Amphipoda) represents an interesting biological model for studying responses of host-parasite interactions to changes in resources quality. This species is generally found in small headwater streams, and is particularly sensitive to environmental changes [START_REF] Maltby | Evaluation of the Gammarus pulex in situ feeding assay as a biomonitor of water quality: robustness, responsiveness, and relevance[END_REF]. It also constitutes a host for many parasitic species ranging from bacteria to macro-parasites such as helminths [START_REF] Grabner | Hidden diversity: parasites of stream arthropods[END_REF]. In particular, several acanthocephalan species, such as Pomphorhynchus laevis, use gammarids as intermediate hosts (Crompton & Nickol, 1985;[START_REF] Kennedy | Ecology of the Acanthocephala[END_REF]. These trophicallytransmitted parasites are well-known for inducing phenotypic modifications in their intermediate hosts, making them more vulnerable to predation by definitive host species [START_REF] Bakker | Parasite-induced changes in behavior and color make Gammarus pulex more prone to fish predation[END_REF]. By this way, parasites increase the probability of completing their life cycle. A few studies have shown that the presence of acanthocephalan parasites might alter the response of gammarids to contaminant exposure, increasing their mortality [START_REF] Brown | Parasitism and host sensitivity to cadmium: An acanthocephalan infection of the freshwater amphipod Gammarus pulex[END_REF] and decreasing their antitoxic defenses [START_REF] Marcogliese | Combined effects of parasites and contaminants on animal health: parasites do matter[END_REF][START_REF] Gismondi | Does the acanthocephalan parasite Polymorphus minutus modify the energy reserves and antitoxic defences of its intermediate host Gammarus roeseli?[END_REF]. More recent studies showed that increased temperature and parasite infection additively reduced gammarids leaf litter consumption (Labaude et al., 2017a).

Finally, the only study to our knowledge starting to investigate the effect of resources quality was carried out by Labaude et al. (2015b), testing the influence of diet protein content on the parasite load and on host behavioral modifications. Their results mainly showed that healthier, well fed-gammarids exhibited higher infection intensity without any significant alteration of hosts' behaviors.

Following the river continuum concept (RCC; [START_REF] Vannote | The river continuum concept[END_REF], headwater streams, where can be found the detritivorous invertebrate G. pulex, are generally described as small streams flowing in forested areas (or at least with a large canopy cover), at least in temperate watersheds from the northern hemisphere. Their functioning is generally related to allochthonous inputs of terrestrial detritus (leaf litter, dead wood, soil leachates…), these detritus representing the main energy and nutrient source at the basis of food webs. Yet, due to their small size and the anthropised surfaces they drain, these streams have long been physically and chemically altered, especially when they are situated in agricultural contexts [START_REF] Moore | Invertebrate biodiversity in agricultural and urban headwater streams: implications for conservation and management[END_REF]. In particular, headwater streams that are generally naturally quite oligotrophic can receive large inputs of nutrients, such as nitrogen and/or phosphorus [START_REF] Carpenter | Nonpoint pollution of surface waters with phosphorus and nitrogen[END_REF][START_REF] Sims | Phosphorus loss in agricultural drainage: Historical perspective and current research[END_REF][START_REF] Peterson | Control of nitrogen export from watersheds by headwater streams[END_REF]. Moreover, a large proportion of these streams have been or are currently submitted to morphological changes as well as a degradation of the riparian corridors [START_REF] Naiman | Riparia: ecology, conservation, and management of streamside communities[END_REF], inducing in turn an increase in instream algal production and a reduction of the relative contribution of allochthonous detritus inputs in comparison with autochthonous primary production [START_REF] Feio | Functional indicators of stream health: a river-basin approach[END_REF]. Thus, headwater stream perturbations should lead to higher elemental quality of resources for detritivorous invertebrates, microbial decomposers being able to immobilize dissolved nutrients in their biomass [START_REF] Cross | Consumer-resource stoichiometry in detritus-based streams[END_REF], but also permit to these invertebrates to access higher amounts of algae, these ones furnishing essential compounds for detritivores' growth and survival [START_REF] Crenier | Minor food sources can play a major role in secondary production in detritus-based ecosystems[END_REF][START_REF] Rollin | Additive effect of calcium depletion and low resource quality on Gammarus fossarum (Crustacea, Amphipoda) life history traits[END_REF].

In this study, we tested the effects of resources quality on the infection rate, parasite load, hosts' survival, and parasite-mediated behavioral traits presumably implied in trophic transmission (refuge use, geotaxis and locomotor activity of the surviving amphipods). Three resources were used for feeding individually G. pulex experimentally infected or not by the acanthocephalan species P. laevis: microbially-conditioned leaf litter without phosphorus input (standard resource, S), microbially-conditioned leaf litter enriched in phosphorus (P), and microbially-conditioned leaf litter without phosphorus input but complemented with additional inputs of benthic diatoms rich in both phosphorus and eicosapentaenoic acid (EPA, D). As reported in [START_REF] Rollin | Additive effect of calcium depletion and low resource quality on Gammarus fossarum (Crustacea, Amphipoda) life history traits[END_REF], we assumed that addition of algal resources to low quality leaf litter will have a greater impact than phosphorus-enriched leaf litter, algae bringing both phosphorus and essential fatty acids. We hypothesized that higher quality resources, regardless of the infection status, will reduce G. pulex mortality (hypothesis 1) and increase their growth (hypothesis 2). Higher quality resources, by increasing the energetic status of hosts, is expected to increase parasite load (hypothesis 3), modify behavior of hosts (hypothesis 4) and increase parasite impacts on hosts' behavioral changes (hypothesis 5).

Materials and Methods

Sampling and maintenance

Sycamore maple (Acer pseudoplatanus, Linnaeus) leaf litter was collected at abscission from the riparian zone of a second-order forested stream located upstream of any direct anthropogenic pollution (La Maix stream, Vosges Mountains, north-eastern France; 7°03′14•2″E; 48°29′24 9″N). This litter is commonly used in our experiments (see [START_REF] Danger | Phosphorus content in detritus controls life-history traits of a detritivore[END_REF][START_REF] Arce-Funck | Stoichiometric constraints modulate impacts of silver contamination on stream detritivores: an experimental test with Gammarus fossarum[END_REF]Rollin et al., 2017) because it is uncontaminated and its physico-chemical characteristics are well described. Sycamore maple litter was dried in the laboratory and stored at room temperature until use.

Water was collected in La Mance stream (6°02′24•4″E; 49°05′11 7″N), another headwater forested stream presenting similar water physico-chemical characteristics compared to G. pulex native stream ones (see below): pH = 8.2, conductivity = 580 µS cm -1 , acid neutralizing capacity (ANC) = 4720 µeq L -1 , Cl -= 13.3 mg L -1 , NO 3 -= 24.4 mg L -1 , SO 4 2-= 31.7 mg L -1 , PO 4 3-< 0.1 mg L -1 , Na + = 6.5 mg L -1 , Ca 2+ = 100 mg L -1 , K + = 0.82 mg L -1 , and Mg 2+ = 5.76 mg L -1 .

Specimens of G. pulex were sampled in April 2015 by kick-sampling technique in a small tributary of the Suzon river (Burgundy, eastern France; 47°24'12.6"N ; 4°52'058.2"E), an unpolluted forested stream. Over the past 20 years, the acanthocephalan parasite P. laevis was never found in this stream, the G. pulex population in place is thus naïve for this parasite (see Labaude et al., 2015b). During the sampling, only males G. pulex were kept because the experimental parasite infection was proved to be more efficient in males than in females [START_REF] Franceschi | The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation[END_REF]. No noticeable infected gammarids were found. The animals were placed in plastic coolers and rapidly transported to the laboratory, where they were sorted to keep only one homogenous size. Individuals were kept in groups of 500 organisms in aerated aquaria (37 x 55 x 10 cm) filled with La Mance water and acclimatized to laboratory conditions (15°C ± 1; 12:12 h light:dark cycle) for 10 days prior to experimental infections.

Sycamore maple leaves were provided ad libitum as food resource.

Parasite eggs were collected from five naturally-parasitized chubs (Squalius cephalus) caught in the Vouges river (Burgundy, eastern France, 47°9'34.36" N; 5°9'2.50" E). Fish were anaesthetized, killed and dissected within 24 h after collection. Female parasites were immediately taken from the intestines of the fish. Parasite eggs were obtained after dissection of ten P. laevis gravid females. The taxonomy of parasites was verified by genetic analyses as described in [START_REF] Franceschi | The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation[END_REF]. Sampled eggs were pooled in La Mance water to obtain a concentrated but homogenous solution (1 mL). The number of eggs by microliter was counted using a microscope on ten replicates.

Experimental design

Six combinations were studied in a fully balanced 3 × 3 factorial design (Fig. 1). Three resource qualities (see 2.3.) were tested cross wise with the three parasite conditions related to laboratory infestation procedure and then egg exposure/infection status.

The three tested resources qualities were i) conditioned maple leaves without P addition (Standard litter, S, low quality treatment) or ii) with P addition (P, medium quality treatment)

or iii) with diatom supplementation using agarose pellets (D; in this case P and PUFA was supplied by diatoms; high quality treatment; see [START_REF] Rollin | Additive effect of calcium depletion and low resource quality on Gammarus fossarum (Crustacea, Amphipoda) life history traits[END_REF].

The three parasite conditions were: a) unexposed gammarids (Ue), b) exposed but uninfected gammarids (Ui), and c) exposed and successfully infected gammarids (I). For statistical analysis Ui and I conditions were also regrouped as exposed condition (E) and then compared to Ue.

Resource treatments

Food resources were prepared in large quantities following the procedure described in [START_REF] Rollin | Additive effect of calcium depletion and low resource quality on Gammarus fossarum (Crustacea, Amphipoda) life history traits[END_REF].

Leaf phosphorus manipulation

To have marked differences in stoichiometric leaves qualities, we selected maple leaves as they contain initially low phosphorus content and exhibit a quite low carbon quality (Hladyz et al., 2009). Dried maple leaves were soaked for a few minutes in deionized water, and 21,000 discs (20 mm in diameter) were then cut using a disc borer while avoiding the main veins. These discs were placed by 200 in fine mesh size bags (mesh: 200 µm; 15 x 25 cm) to avoid macroinvertebrate access. Bags were transferred in La Maix stream during 7 days for conditioning by microorganisms such as hyphomycetes and bacteria.

In the laboratory, discs were then rinsed using La Maix water and randomly introduced into 105 Erlenmeyer flasks (500 mL, 200 discs per flask). Then, we added 200 mL of sterile medium deprived from phosphorus in each flask. This treatment served as the standard food treatment (low resource quality, S). In 35 randomly selected flasks, we introduced 4.64 mL of KH 2 P0 4 at 8.71 g phosphorus.L -1 , which served as the phosphorus implemented food treatment (high quality resource 1, P; protocol fully described in [START_REF] Danger | Phosphorus content in detritus controls life-history traits of a detritivore[END_REF]. Discs were incubated in the flasks for 4 days in the dark at 14°C on an orbital shaker, then sorted, rinsed with deionized water, and stored at -20°C until use.

Supplementation of diatoms in agarose pellets

An axenic strain of the diatom Nitzschia palea (reference CPCC 160; high amount of EPA) was grown during 2.5 weeks in four 5 L-Erlenmeyer flasks filled with 4 L of Combo medium (Kilham et al., 1998). This culture was maintained at 20°C in a temperaturecontrolled chamber (50 µE, light/dark 16:8) under continuous shaking (150 rpm). Cells were harvested by centrifugation (4,000 g, 10 minutes) at the end of the exponential growth phase

(2.5 weeks) and were freeze-dried and stored at -20°C until use.

Food source pellets were prepared using low gelling temperature agarose (Sigma A9414) as a nutrient-free matrix, as fully described in [START_REF] Crenier | Minor food sources can play a major role in secondary production in detritus-based ecosystems[END_REF]. Briefly, agarose was dissolved in glass bottles containing demineralized water (2%), heated in a microwave, and placed in an agitated thermostatic water bath at 38°C. Risk of deterioration of fatty acids due to excessive temperature was limited due to the low gelling point of agarose and the low temperature applied. A mixture of 25 mg of diatoms and 1.6 ml of agarose solution was introduced in a 2 ml Eppendorf® tube (to obtain a 50% carbon supply), homogenized using a vortex and placed in the thermostatic bath until use to avoid agarose gelling. The pellets were formed by injecting the homogenate in the holes of a Plexiglas® mold composed of 40 holes

(5 mm diameter x 2 mm high: 39.27 mm 3 ). The pellets were unmolded after a few minutes and were kept at -20°C in Petri dishes until use. Pellets were provided along with unmanipulated leaves (S) to constitute a second high quality treatment (D), which was the higher resource quality treatment. These pellets supplied EPA, phosphorus, and other elements potentially of interest for organisms' development.

Resource content analysis

Four batches of 10 frozen leaf discs per treatment (S, P, see 2. Technologies TM 6 850 gas chromatograph. FAMEs were identified by comparing retention times with those obtained from Supelco ® and laboratory standards, and quantified against internal standards (13:0).

Infection procedure of G. pulex

Experimental infections were performed following the procedure described in [START_REF] Franceschi | The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation[END_REF] and Labaude et al. (2015b). First, gammarids were starved during 24h in individual 60 ml glass dishes (15°C ± 1; 12:12 h light:dark cycle) to avoid cannibalism and maximize gammarids feeding during infection procedure. Second, gammarids were pooled by two in glass dishes (6 cm diameter) containing 50 mL of La Mance water at 15 ± 1°C. Then, a conditioned maple leaf disk (S, see 2.3.1.), on which 100 P. laevis eggs were previously deposited, was provided in each dish. Gammarids were allowed to feed on these disks for 48 h, thus initiating the infection procedure, i.e. G. pulex exposure (Exposed, E). In the same experimental conditions, a control group, consisting of unexposed organisms (S leaf disc but without parasite eggs, Ue), was performed to control for potential confounding effects of infection procedure on gammarid endpoints. Overall, 1014 gammarids were exposed to parasite eggs and 330 were unexposed in the same conditions.

After this infection step, living gammarids were rinsed and were placed individually in new dishes in the same experimental conditions. Then, the three resource quality treatments were randomly allocated for exposed and unexposed gammarids (338 and 110 gammarids per food quality treatment, respectively; see table 1). Dead organisms (n=62) and gammarids naturally infected by other parasite species (n=11) during the infection procedure were removed from the analyses (differences between the two first column in Table 1).

Experimental setup

After the infection period, male gammarids were photographed for initial size measurement at the beginning of the experiment (see 2.6.1.; ANOVA; no significant size differences between treatments were observed; Mean size: 10.39 ± 0.11 mm; p > 0.05). The experiment was conducted for 110 days in a natural unpolluted water at 15 ± 1°C under a 12:12 h light:dark cycle, which represents conditions close to gammarids optimum for growth and survival. Each replicate consisted of a 60-mL glass dish containing 50 mL of La Mance water, a male G. pulex (unexposed or exposed to parasite eggs, and then successfully infected or not), and the corresponding food resource (1 disc of maple leaf with or without phosphorus addition and with or without agar pellet). To limit chemical variations and to ensure that G.

pulex were fed ad libitum, water and food resources were renewed weekly.

Six weeks after the first infected G. pulex was observed (day 42), all dead gammarids were dissected under a stereomicroscope to verify their infection status. Gammarids in which parasites failed to develop are referred as uninfected (Ui) in the following parts. After 110 days, gammarids were photographed prior to the behavioral assays. This 110-day period appeared to be a good compromise between measuring survival for a long enough time and getting enough animals to perform behavioral tests. After behavioral assays, gammarids were sacrificed and dissected under a stereomicroscope to determine their infection status (infected or uninfected) and their parasite load.

Endpoints

Gammarids survival

The number of dead gammarids was recorded daily. The date of death of each individual was noted and dead gammarids were immediately photographed for size assessment (see 2.6.2.) and dissected to assess the presence and the number of parasites (see 2.6.4.).

Gammarids size and growth rate

Gammarids were photographed under a stereomicroscope at the day they were found dead or at the day 110 (end of the experiment). The height of their fourth coxal plate was measured using SigmaScan Image Analysis Version 5.000 (SPSS Inc, Chicago, IL, USA). This metric is a reliable proxy for gammarids size [START_REF] Bollache | The influence of micro-habitat segregation on size assortative pairing in Gammarus pulex (L.) (Crustacea, Amphipoda)[END_REF] and is widely used. To determine gammarids body length (mm), we used the allometric relation established by [START_REF] Labaude | Effect of the environment on the interaction between gammarids (Crustacea : Amphipoda) and their manipulative acanthocephalan parasites[END_REF] for males G. pulex coming from the same sampling site [body length (mm) = 4.33 × fourth coxal plate length (mm); R 2 = 0.61, p < 0.0001].

Individuals growth rates (mm.mm.day -1 ) were calculated by dividing the difference between the final size and the initial size by the initial size multiplied by the number of days between the two measures.

Behavioral assays

The day after the end of the experiment, we assessed shelter use of each living gammarids. The second day, we measured geotaxis of the same gammarids, followed the third day by locomotor activity. For all of behavioral assays, blind recordings were ensured by labelling testing device with a number giving no idea about the treatment to which gammarids were belonging. In order to allow gammarids to choose their position without having to swim, the inner walls of the cylinders were covered with nylon mesh, making it possible to cling. This aspect was important since the parasite is able to alter both the swimming and the clinging behaviors of gammarids [START_REF] Bauer | The acanthocephalan parasite Polymorphus minutus alters the geotactic and clinging behaviours of two sympatric amphipod hosts: the native Gammarus pulex and the invasive Gammarus roeseli[END_REF]. Each cylinder was placed at 15°C in a temperature-controlled chamber in which the light came only horizontally to avoid any confounding phototactic reaction. After 3 minutes of acclimatization, the position of gammarids was recorded every 15 s for 5 min and scored from 1 (bottom) to 5 (top compartment). The cumulated geotaxis score therefore ranged from 20 (always in the bottom zone) to 100 (always in the top zone) for each individual.

The locomotor activity was assessed following the method used by [START_REF] Sornom | Effect of gender on physiological and behavioural responses of Gammarus roeseli (Crustacea Amphipoda) to salinity and temperature[END_REF].

This method usually measures the proportion of time spent moving. In total, locomotor activity assays were performed on 57 unexposed G. pulex (Ue; 14, 22 and 21 for S, P and D resource treatment, respectively) and 13 infected G. pulex (I; 1, 6 and 6 for S, P and D, respectively). We placed the gammarids individually in 8 cm diameter Petri dishes open to the air and filled with La Mance water. Petri dishes were then placed in a hermetic box linked to a digital video camera. After 5 minutes of acclimatization, individuals' behavior was videorecorded under red light at 15 frames per second for 5 min. Locomotor activity was scored as the proportion of time swimming. Below 15 mm.s -1 , moving gammarids were considered crawling rather than swimming.

Prevalence and parasite load

All gammarids were dissected under stereomicroscope (×6 to ×40 magnification) either the day individuals were found dead or at the end of the experiment. This step permitted to remove gammarids infected by other parasites than P. laevis, define the status of exposed G. pulex (infected, uninfected), the parasite load (number of parasites in one G.

pulex) and the prevalence of parasite (% of infected G. pulex) in each experimental condition.

Statistical analyses

The first infected gammarid was observed at day 47. Thus, two datasets were used for survival rate and parasite load statistical analysis to integrate or not the delay of infection in the analysis (from day 0 to 110 vs from day 47 to 110). Indeed, before 47 days of experiment, it was not always possible to identify if gammarids were infested or not, due to the too low development of parasites. For other endpoints, only the results obtained at the end of the experiment were considered.

Statistical analyses were conducted using R software (R Core Team, 2016) to the 5% level of significance. Survival data were analyzed by Kaplan-Meier survival curves and logrank tests were performed to compare curves and determine the effects of infection status and resource quality on G. pulex survival rate throughout the experiment: from day 0 to 110

[difference between exposed and unexposed] and from day 47 to 110 [difference between infected and uninfected. In the last case, mortality before days 47 (first infection detected)

was not considered]. As the number of infected gammarids was very low, log-rank tests were also performed (day 47 to 110) by pooling infected and uninfected gammarids data or data of gammarids fed on S, P and D resources to better assess the global effect of food resource or effect of infection status, respectively. Bonferroni adjustments were performed on log-rank tests to determine effects of resource quality and infection status on survival.

Due to the extremely low survival in infected-S treatment (n=1), only main effects were testable for gammarids growth size and behavioral responses. Influence of food quality and infection status on G. pulex growth was tested using Kruskal-Wallis tests, and post hoc tests were performed on main effects (Mann-Whitney U-test and Bonferroni adjustment).

A test for proportion comparison permitting the calculation of 95% confidence intervals was used to assess the effect of resource quality on the proportion of infected gammarids.

Significance between differences were assessed using confidence intervals. A multiple

Poisson regression model was used to investigate the effect of resource quality on individual parasite load. In the analyses, the S resource treatment was considered as reference levels.

Kruskal-Wallis tests were used to assess the effect of resource quality on each behavior score (geotaxis, shelter use, locomotor activity). Cliff's deltas [START_REF] Cliff | Ordinal methods for behavioral data analysis[END_REF] were calculated to evaluate the behavior score differences of infected (I) and unexposed (Ue) organisms fed with the different resource qualities (S, P, D). Then, effect sizes for each score between unexposed (Ue) and infected (I) organisms were also compared for P and D resource qualities (not for S due to the too low number of surviving organisms). Cliff's delta, robust to non-normally distributed data, is a scale-less parameter ranging from -1 to 1. It is used to represent the size of the effect and the direction of this effect size difference. Significance between differences was assessed using confidence intervals. The R-package 'orddom' (version 3.1) was used to calculate Cliff's deltas medians and 95% confidence intervals.

Results

Resource compositions

Low resource quality leaves (S) contained 0.36 ± 0.08 mg phosphorus.g -1 and manipulated leaves (P) contained 1.24 ± 0.16 mg phosphorus.g -1 . The dominant fatty acids in conditioned leaves (S or P) were palmitic acid (16:0), oleic acid (18:1ω9), linoleic acid (18:2ω6), and α-linolenic acid (18:3ω3), whereas long chain PUFAs (number of Cs ≥ 20)

were totally lacking from these resources.

Agarose pellets contained 1.95 ± 0.58 mg phosphorus.g -1 . In diatoms the major fatty acids were palmitic acid (16:0), palmitoleic acid (16:1ω7), and EPA (20:5ω3). As mentioned in [START_REF] Rollin | Additive effect of calcium depletion and low resource quality on Gammarus fossarum (Crustacea, Amphipoda) life history traits[END_REF], these compounds accounted for more than 65% of total fatty acids (16:0 = 28.2%, 16:1ω7 = 21%, and 20:5ω3 = 16.9%). Docosahexaenoic acid (22:6ω3) represented 2.2% of diatoms fatty acids.

G. pulex survival

After 47 days of exposure (day of the first observed parasite), the survival of gammarids was high, ranging from 81.7 % in exposed (E; Ui+I)-P treatment to 96 % in Ue-D treatment (Table 1). At 110 days, survival rates were low, ranging between 15.4 % in Ue-S treatment and 33.9 % in E-D treatment. The highest G. pulex survival was observed when organisms fed on the highest resource qualities (D>P>S), regardless of the infection status (Table 1).

The Log-rank global comparison performed on G. pulex exposed or not to P. laevis eggs (Fig 2a ., day 0 to 110) and fed on one of the three resource qualities reports highly significant differences in gammarid survival rates (Chi 2 = 30.9, d.f. = 5, p = 9.68 x 10 -6 ). Two by two logrank tests with Bonferroni adjustment showed that survival in E-D and Ue-D treatments (highest quality resource) was significantly higher than survival in E-S and Ue-S treatments (the lower quality resource) [E-D vs E-S: Chi 2 = 21.6, d.f. = 1, p = 3.82 x 10 -6 ; E-D vs Ue-S:

Chi 2 = 9, d.f. = 1, p = 0.0027; Ue-D vs Ue-S: Chi 2 = 11, d.f. = 1, p = 9 x 10 -4 ; Ue-D vs E-S:

Chi 2 = 17.3, d.f. = 1, p = 3.21 x 10 The effect of food resource was clearly significant, but due to a low infected gammarids number, the effect of infection was not observed using the 2 by 2 log-rank. When log-rank was performed on pooled infection status data (infected and uninfected; day 47 to 110), a significant global effect of food treatment was observed (Chi 2 = 16.9, d.f. = 2, p = 2.19 x 10 -4 ) with gammarid fed with S resource showing significant lower survival compared to those fed with P (Chi 2 = 6.9, d.f. = 1, p = 0.00865) and D resources (Chi 2 = 16.4, d.f. = 1, p = 5.25 x 10 - 5 ). In the same way, when log-rank tests were performed on pooled food resource data (S, P and D; day 47 to 110), a significant global effect of infection status was reported (Chi 2 = 10.9, d.f. = 2, p = 0.00433), with uninfected gammarids showing significantly higher survival compared to those infected by P. laevis (Chi 2 = 9.4, d.f. = 1, p = 2.155 x 10 -3 ). Thus, survival was influenced by both resource quality and infection status, but no interactive effect was observed. No global survival differences were reported between unexposed and uninfected gammarids (Chi 2 = 3.4, d.f. = 1, p = 0.0638) showing the absence of infestation procedure impact.

G. pulex growth

Resource quality had a significant effect on G. pulex growth (Kruskal-Wallis; Chi 2 = 140.69, d.f. = 2, p = 2.2 x 10 -16 ; Fig. 3). Two by two Mann Whitney U-test with Bonferroni adjustment revealed that size growth of G. pulex fed with D resource was significantly higher than that of gammarids fed with P and S resources (D vs P: Chi 2 = 2343.5, d.f. = 1, p = 2.2 x 10 -16 ; D vs S: Chi 2 = 1398, d.f. = 1, p = 2.2 x 10 -16 ) and that the growth of G. pulex fed with P resource was significantly higher than that of gammarids fed with S resource (Chi 2 = 2680, d.f. = 1, p = 4.4 x 10 -8 ). On average, compared to S treatment, gammarids growth was increased by 17 and 56.4 % in individuals fed with P and D resource, respectively.

Infection status also had a significant effect on G. pulex growth (Kruskal-Wallis, resource quality: Chi 2 = 9.66, d.f. = 2, p = 0.008). The growth of infected gammarids was similar regardless of the resource quality. Two by two Mann Whitney U-test with Bonferroni adjustment revealed that the growth of G. pulex infected by P. laevis was significantly lower than that of uninfected gammarids (I vs Ui: Chi 2 = 3703, d.f. = 1, p = 0.002). On average, compared to uninfected gammarids, size growth of infected gammarids was reduced by 20.3 %.

Prevalence and parasite load

G. pulex infection success by P. laevis was significantly affected by resource quality, both when considering all gammarids (dead and alive) taken after 47 days (Chi 2 = 47.29, d.f. = 2, p = 5.38 x 10 -11 , Fig. 4a) and those still alive at the final date (110 days; Chi 2 = 6.21, d.f. = 2, p = 0.045, Fig. 4b), the number of infected G. pulex increasing while increasing resource quality.

Among the gammarids exposed to P. laevis eggs, 2.7 % [1.2-5.2], 11.4 % [8.2-14.4] and 20.1 % [15.8-25.0] of G. pulex (mean % [95% confidence interval]) were infected and alive after 47 days of feeding on S, P, and D resources, respectively (n=10, 38, and 63; Table 1). Based on the 95% confidence intervals, it can be deduced that infection success significantly differed between the three resource qualities. At 110 days, only 1.7 % [0.04-8.5],

7.2 % [2.6-15] and 20.1 % [7.1-21.4] of gammarids, representing 1, 7 and 14 individuals, were alive and infected in S, P and D treatment, respectively (Table 1).

Among infected G. pulex, amphipods fed with D resource had a significantly higher P.

laevis load when considering all individuals, dead or not (day 47 to 110; S as reference level; p = 2.73 x 10 -13 , Fig. 4c), or only the surviving ones (day 110; S as reference level; p = 0.012, Fig. 4d). P resource also significantly increased P. laevis load in individual gammarid but only when all individuals, dead or not, were considered (day 47 to 110; S as reference level; p = 4.13 x 10 -6 , Fig. 4c). Considering all individuals, the relative proportion of infected gammarids with only one P. laevis was reduced by a factor of 1.4 between S and P resource and by a factor of 1.9 between S and D resources (by 2 and by 2.6 at final time, but only one organism was present in infected-S treatment).

Behavior

Resource quality had a marginally significant effect on G. pulex geotaxis score (Kruskal-Wallis; Chi 2 = 6.15, d.f. = 2, p = 0.046; Fig. 5a), but not on shelter use (Chi 2 = 1.53, d.f. = 2, p = 0.47; Fig. 5b) or locomotor activity (Chi 2 = 2.69, d.f. = 2, p = 0.26; Fig. 5c). Two by two Mann Whitney U-test with Bonferroni adjustment showed that mean geotaxis scores among resource treatments were similar (mean values of 28.9, 36.8, and 38.6 for S, P, and D, respectively), while Cliff's delta revealed a significantly higher geotaxis score in Ue-D and Ue-P compared to Ue-S treatment (Fig. 6a.). Unexposed gammarids fed on higher quality resource were significantly higher in the water column.

Compared to unexposed G. pulex, those infected with P. laevis showed a significantly higher geotaxis score (Mann-Whitney; U = 338, d.f. = 1, p = 1.652 x 10 -4 ; in mean 32.7 vs 53.6; Fig. 5a), a significantly higher shelter use score (Mann-Whitney; U = 505.5, d.f. = 1, p = 0.0244; in mean 6.7 vs 10.3; Fig. 5b), but no differences were reported for locomotor activity (Mann-Whitney; U = 340, d.f. = 1, p = 0.6504; in mean 0.364 vs 0.378; Fig. 5b). Infected gammarids thus tended to be higher in the water column and more often outside of the refuge.

However, when considering Cliff's delta approach, no significant differences were reported between treatments.

Discussion

To date, only few studies evaluated the effect of stoichiometric quality of the resource, excluding confounding factors (e.g. changes in microbial biomass, alteration of detrital carbon quality), on the survival and life history traits of a detritivorous species [START_REF] Danger | Phosphorus content in detritus controls life-history traits of a detritivore[END_REF][START_REF] Arce-Funck | Stoichiometric constraints modulate impacts of silver contamination on stream detritivores: an experimental test with Gammarus fossarum[END_REF][START_REF] Rollin | Additive effect of calcium depletion and low resource quality on Gammarus fossarum (Crustacea, Amphipoda) life history traits[END_REF]. Similarly, studies on the effect of stoichiometric quality of the resource on parasitic infection are scarce and did not concerned detritivorous hosts (Frost andElser, 2008, Schlotz et al., 2013), Stephens et al., 2016).

In the present study, comparison of S (microbial conditioned maple leaves) and P (phosphorus-manipulated conditioned maple leaves) resources treatment allowed investigating stoichiometric effects due to the higher phosphorus content in food resource [START_REF] Danger | Phosphorus content in detritus controls life-history traits of a detritivore[END_REF]. In addition, a supplementation of leaf litter with diatom pellets (D) was expected to greatly increase resource quality for consumers. Indeed, diatoms are easier to assimilate than detritus, they show high elemental quality (higher phosphorus contents than leaf litter), and contains high EPA concentration [START_REF] Crenier | Minor food sources can play a major role in secondary production in detritus-based ecosystems[END_REF].

Resource quality was the main driver of G. pulex survival rates, infected or not by P.

laevis. Higher survival was related to higher quality of resources consumed by G. pulex (S<P<D). Higher growth rates were also reported for G. pulex fed on higher resource quality.

These results are in accordance with several studies reporting a beneficial effect on aquatic organisms' survival and growth rate of resources containing high phosphorus or EPA rates (Daphnia: [START_REF] Makino | Evidence of phosphorus-limited individual and population growth of Daphnia in a Canadian Shield lake[END_REF][START_REF] Masclaux | Combined effects of food quality and temperature on somatic growth and reproduction of two freshwater cladocerans[END_REF][START_REF] Schlotz | The potential of dietary polyunsaturated fatty acids to modulate eicosanoid synthesis and reproduction in Daphnia magna: A gene expression approach[END_REF][START_REF] Prater | Effects of calcium and phosphorus limitation on the nutritional ecophysiology of Daphnia[END_REF] Oncorhynchus mykiss: [START_REF] Lellis | Effects of phase-feeding dietary phosphorus on survival, growth, and processing characteristics of rainbow trout Oncorhynchus mykiss[END_REF]Gammarus: Kraufvelin et al., 2006;[START_REF] Danger | Phosphorus content in detritus controls life-history traits of a detritivore[END_REF][START_REF] Gergs | Dietary supply with essential lipids affects growth and survival of the amphipod Gammarus roeselii[END_REF][START_REF] Crenier | Minor food sources can play a major role in secondary production in detritus-based ecosystems[END_REF]Rollin et al., 2017). According to Rollin et al.

(2018), higher G. pulex growth rate when fed on higher resource quality was linked to a higher molt frequency. This is in accordance with the growth rate hypothesis stating that growth requires high phosphorus amounts, phosphorus being involved in the synthesis of phosphorus-rich RNA necessary for ensuring organisms' growth [START_REF] Schade | Stoichiometric tracking of soil nutrients by a desert insect herbivore[END_REF].

Beneficial effect of PUFA on gammarids' growth might be due to their inability to maintain their PUFA content and/or to synthesize these essential compounds [START_REF] Crenier | Minor food sources can play a major role in secondary production in detritus-based ecosystems[END_REF].

In the present study, G. pulex infected by P. laevis showed lower survival and lower growth rates than those unexposed and uninfected. These results are in accordance with previous studies [START_REF] Lafferty | Parasites in food webs: The ultimate missing links[END_REF][START_REF] Aalto | Food stoichiometry affects the outcome of Daphnia-parasite interaction[END_REF]Labaude et al., 2015b;[START_REF] Labaude | Effect of the environment on the interaction between gammarids (Crustacea : Amphipoda) and their manipulative acanthocephalan parasites[END_REF]. Growth reduction could be related to the energetic costs of harboring a parasite. Crustaceans infected by acanthocephalan parasites showed significant modifications in their energetic reserves [START_REF] Plaistow | The effect of the acanthocephalan parasite Pomphorhynchus laevis on the lipid and glycogen content of its intermediate host Gammarus pulex[END_REF][START_REF] Gismondi | Does the acanthocephalan parasite Polymorphus minutus modify the energy reserves and antitoxic defences of its intermediate host Gammarus roeseli?[END_REF][START_REF] Caddigan | Intra-population variation in behavior modification by the acanthocephalan Acanthocephalus dirus: are differences mediated by host condition?[END_REF], and isopods infected by acanthocephalans were reported to allocate about 21% of their energy production to parasite growth, to the detriment of their own reproduction [START_REF] Lettini | The energetic cost of parasitism in isopods[END_REF].

Leaving enough resources to their hosts might be a strategy selected in parasites, allowing them to manipulate host behaviors [START_REF] Maure | How much energy should manipulative parasites leave to their hosts to ensure altered behaviours[END_REF]. According to this hypothesis, when the host condition is lowered, a lower parasite exploitation is expected. In the present study both low resource quality (S) and infection with P. laevis significantly reduced G. pulex survival, but no clear interactive effects were identified. Survival was systematically lowered for infected G. pulex compared to uninfected organisms whatever the resource quality. In addition, the growth of infected gammarids was similar regardless of the resource quality. Therefore, our results did not confirmed the hypothesis of [START_REF] Maure | How much energy should manipulative parasites leave to their hosts to ensure altered behaviours[END_REF], and were in accordance with Labaude et al. (2015b). Similarly, Frost et al. (2008) showed that low phosphorus content in resource increased deleterious effects of bacterial infection on Daphnia reproduction.

One of the important results of our study concerns the impacts food stoichiometric quality had on G. pulex infection by the acanthocephalan species P. laevis: high resources qualities were associated to higher prevalences. Several studies have shown that high nutrient availabilities in ecosystems tended to increase the prevalence of parasites [START_REF] Johnson | Linking environmental nutrient enrichment and disease emergence in humans and wildlife[END_REF][START_REF] Altman | Large-scale spatial variation in parasite communities influenced by anthropogenic factors[END_REF]. However, most of these studies mainly considered resources quantity, rarely resources quality. Insertion ici de la dernière partie de ce paragraphe? Our results thus provide an argument for a positive effect of the quality of resources on a parasitic infection. This in in line with results of Frost and Elser (2008), showing that the proportion of Daphnia infected, as well as the number of parasites per Daphnia, were largely increased when organisms fed on low Carbon/Phosphorus resources, or those of Stephens et al. (2016), that showed on amphibian parasites that higher litter quality does not decrease susceptibility to infection. Therefore, in these cases, higher amounts of energy in the host might permit a better development of parasites, rather than allowing the host to increase its energy, allowing better defenses [START_REF] Arce-Funck | Stoichiometric constraints modulate impacts of silver contamination on stream detritivores: an experimental test with Gammarus fossarum[END_REF], Stephens et al., 2016). Another interesting result is the increase in the parasite load (number of P. laevis per gammarid) for organisms fed with the highest quality resources (P and D). This result suggests that the higher energy contents of gammarids fed with high quality resources, already observed in Arce [START_REF] Arce-Funck | Stoichiometric constraints modulate impacts of silver contamination on stream detritivores: an experimental test with Gammarus fossarum[END_REF], favored several parasites to co-exist in a single individual through reducing competition between parasites. Interestingly, [START_REF] Schlotz | Dietary supply with polyunsaturated fatty acids and resulting maternal effects influence host-parasite interactions[END_REF] also observed that infected daphnids by the parasite Pasteuria ramosa showed the highest spore load per animal when fed a high quality food. Even if the number of infected organisms at the end of the experiment preclude strong statistical testing, the number of parasites per gammarid seemed unrelated to higher mortality, the proportion of multi-infected gammarids remaining unchanged throughout the experiment. This result is in accordance with the results of [START_REF] Franceschi | The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation[END_REF] who did not observe any effect of infection intensity on host survival. Finally, we showed that diatom supplementation led to significantly higher infection rates than phosphorus-enriched resources alone, suggesting that in ecosystems, consumption of such a high quality resource, bringing both nutrients and PUFA, might strongly impact parasites development, at least more than ingestion of leaf litter conditioned in streams with high nutrient loads. This part is particularly important in a global context of degradation of riparian vegetation and canopy opening in the vicinity of streams [START_REF] Hladyz | Stream ecosystem functioning in an agricultural landscape: the importance of terrestrial-aquatic linkages[END_REF].

Despite the important effects of resources quality on host survival and growth, as well as on parasite prevalence and infection intensity, our study did not reveal clear impacts of resources quality on the modulation of parasites-induced behavioral changes of hosts. As expected, after 110 days of experiment, P. laevis infection led to changes in hosts' geotaxis and shelter use. Behavioral changes have long been described in this host-parasite interaction [START_REF] Bakker | Parasite-induced changes in behavior and color make Gammarus pulex more prone to fish predation[END_REF][START_REF] Bauer | The acanthocephalan parasite Polymorphus minutus alters the geotactic and clinging behaviours of two sympatric amphipod hosts: the native Gammarus pulex and the invasive Gammarus roeseli[END_REF][START_REF] Lagrue | Modification of the hosts' behavior by a parasite: field evidence for adaptive manipulation[END_REF]. These changes, according to the manipulation hypothesis [START_REF] Holmes | Modification of intermediate host behaviour by parasites[END_REF], predispose manipulated intermediate hosts to be preyed upon by definitive hosts, i.e. a fish. Such behavioral changes are mainly related to parasite-induced changes in hosts' neurotransmitters production [START_REF] Tain | Altered host behaviour and brain serotonergic activity caused by acanthocephalans: evidence for specificity[END_REF], Perrot-Minnot et al., 2014). [START_REF] Maure | How much energy should manipulative parasites leave to their hosts to ensure altered behaviours[END_REF] proposed the "host energetic resource constraint hypothesis" (HERC) stating that a largely overlooked part of infected hosts' energy might be used during the behavioral manipulation of the host. Following this hypothesis, higher resources quality should have permitted the parasite to amplify the behavioral manipulation of its host. The absence of such effect suggests that HERC is not applying in gammarids infected with P. laevis. However, this result must be taken cautiously due to the very low statistical power generated by the very low survival of infected gammarids fed with standard food resources. Results of [START_REF] Franceschi | Co-variation between the intensity of behavioural manipulation and parasite development time in an acanthocephalan-amphipod system[END_REF] suggested a trade-off between P. laevis growth rate and behavioral manipulation. However, working on the same host and parasite species, Labaude et al. (2015b) did not report any effect of resource quality on parasite growth.

Finally, while resources quality had no amplification effect on parasite-mediated host manipulation, resource quality had a small but significant effect on the geotaxis of uninfected behavior following increased food quality, the observed effect on geotaxis probably relies on a higher energetic content of well-fed organisms.

To sum-up, this experiment permitted to highlight that higher quality resources, regardless of the infection status, reduced G. pulex mortality (hypothesis 1 in the introduction), while only uninfected gammarids exhibited higher growth rates when fed with high quality resources (partial validation of hypothesis 2). In addition, higher quality resource increased parasites' infection rates (verifying hypothesis 3). While slightly modifying the geotaxis behavior of uninfected organisms (partially verifying hypothesis 4), resources quality did not modulate parasites impacts on hosts' behavior (rejecting hypothesis 5). Finally, for most parameters investigated, consumption of algal resources had a greater impact than phosphorus-enriched leaf litter. Our experiment thus confirms that in addition to changes in resource quantity, changes in resources quality might play a major role in the currently observed development of parasites. This result stresses the need for future research to investigate in natura the relationships between resources availability, resources quality and parasites prevalence. In particular, it might be important to verify if rivers with higher algal production (e.g. rivers with open canopies; [START_REF] Feio | Functional indicators of stream health: a river-basin approach[END_REF] exhibit higher parasite prevalence. Moreover, since all algal groups do not have the same biochemical quality (e.g.

chlorophytes or cyanobacteria lack EPA, [START_REF] Bec | How well can the fatty acid content of lake seston be predicted from its taxonomic composition?[END_REF], and since diatoms are progressively replaced by chlorophytes or cyanobacteria along nutrient load gradients [START_REF] Leflaive | Community structure and nutrient level control the tolerance of autotrophic biofilm to silver contamination[END_REF], stream eutrophication might lead first to increased parasite prevalence until a decline related to shifts in algal communities. Finally, it would be interesting to evaluate if parasites could modify the host feeding behavior (food selection) toward food with high Phosphorus/algal contents, to allow them a better development. In this context, using the conceptual frameworks of trophic ecology, such as ecological stoichiometry [START_REF] Bernot | Ecological stoichiometry for parasitologists[END_REF]) might permit to understand in more depth the host-parasite interaction in ecosystems, and the impact the anthropic-driven changes might have on these interactions.

Such research might be especially important when considering the importance of parasites in ecological processes, and, in fine, on ecosystem services (Lafferty and Kuris, 2012).

  The sheltering behavior of individual gammarids was recorded by quantifying refuge use according to[START_REF] Dianne | Parasite-induced alteration of plastic response to predation threat: increased refuge use but lower food intake in Gammarus pulex infected with the acanthocephalan Pomphorhynchus laevis[END_REF]. Sheltering behavior assays were performed on 75 unexposed G. pulex (Ue; 15, 31 and 29 for S, P, and D resource treatments, respectively) and 20 infected G. pulex (I; 1, 6, and 13 for S, P and D, respectively). Gammarids were individually placed in boxes (10.5 × 16 cm) filled with 250 mL of La Mance water. At one extremity, each box was containing an opaque refuge, consisting of half a saucer terracotta pot (8.5 cm of diameter) with a 1 cm 2 hole in the convex part. After 5 minutes of acclimatization, the gammarid position was recorded every 3 min during 1 h and scored as 1 when outside or 0 when inside the refuge. The cumulated score of shelter use therefore ranged from 0 (always inside the refuge) to 20 (always outside) for each individual.Geotaxis was estimated as the average vertical position of individuals in the water column according toLabaude et al. (2017b). In total, geotaxis assays were performed on 75 unexposed G. pulex (Ue; 15, 31 and 29 for S, P and D resource treatment, respectively) and 20 infected G. pulex (I; 1, 6 and 13 for S, P and D, respectively). After 110 days of exposure, gammarids were individually placed in a 500 ml-graduated cylinders (high: 35 cm; diameter: 6 cm) vertically divided in five zones of equal height, filled with 250 mL of La Mance water.

  day 47 to 110) fed with one of the three resource qualities also reports highly significant differences in gammarid survival rates (Chi2 = 32.08, d.f. = 5, p = 4.09 x 10 -6 ). Two by two log-rank test with Bonferroni adjustment showed that survival in Ui-S treatment was significantly lower than survival in Ui-P and Ui-D treatments (Ui-S vs Ui-P: Chi 2 = 9.4, d.f. = 1, p = 0.0022; Ui-S vs Ui-D: Chi 2 = 20.1, d.f. = 1, p = 7.33 x 10 -6 ) and that Ui-D treatment had significantly higher survival than I-P and I-D treatments (Ui-D vs I-P : Chi 2 = 12.2, d.f. = 1, p = 4.8 x 10 -4 ; Ui-D vs I-D: Chi 2 = 8, d.f. = 1, p = 4.64 x 10 -3 ). No other differences were found.

  gammarids. As proposed by Arce Funck et al. (2016), who observed a change in (another)

  Perkin Elmer AD6 Autobalance, Perkin Elmer Corp., USA). A CHN elemental analyzer (Carlo Erba NA2100, Thermo Quest CE International, Milan, Italy) was used to quantify carbon and nitrogen contents in resources. Leaves and pellets were burned at 550°C for 12 h,

	solubilized in 10 µl of suprapure HNO 3, and 5 mL of nanopure water were added. Phosphorus
	content was quantified by spectrophotometry.
	Pooled resource material (leaves, diatoms; n = 5) were used to perform fatty acid
	analyses. According to the method proposed by Folch et al. (1957), a chloroform/methanol
	solution was two times used to extract lipids. Then, fatty acids were converted into fatty acids
	methyl-esters (FAMEs) by acid catalyzed transesterification and analyzed on an Agilent

3.1.)

, and four agarose pellets were freeze-dried, ground, and weighed to the nearest 0.001 mg on a microbalance (

  -5 ]. P resource led to intermediate but not significantly different survival.

Among exposed gammarids, between day 47 and day 110, the survival rate ranged from 10 % in I-S treatment to 41.8% in Ui-D treatment. Infected gammarids exhibited a significantly lower survival than unexposed and uninfected organisms (S: 10 % vs 17 and 24.9%; S: 18.4 % vs 33 and 36.7%; S: 22.2 % vs 30.6 and 41.8%; Table

1

).

The Log-rank global comparison performed on G. pulex infected or not by P. laevis

(Fig 2b,

Acknowledgments

This work was supported by the CPER Lorraine-ZAM (Contrat Projet Etat Région Lorraine, Zone Atelier Moselle) and the French National Research Agency (ANR-13-BSV7-0004-01/MULTISTRESS). We are also grateful to P. Rousselle for conducting chemical analyses.