Alexandre Bauer
email: alexandre.bauer@u-bourgogne.fr

Lucie Develay

Sébastien Motreuil

Maria Teixeira

Nelly Debrosse

Thierry Rigaud

Experimental infections reveal differences in specificity, development time and virulence between the acanthocephalan parasite Pomphorhynchus tereticollis and its sympatric counterpart P. laevis, in two cryptic species of Gammarus fossarum

Keywords: experimental infection, Gammarus fossarum, Gammarus pulex, host specificity, infectivity, multi-host parasite, Pomphorhynchus laevis, Pomphorhynchus tereticollis, prevalence Methods

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Multi-host parasites are facing hosts of different qualities, which can contribute unequally to their transmission [START_REF] Rigaud | Parasite and host assemblages: embracing the reality will improve our knowledge of parasite transmission and virulence[END_REF]. Key hosts, contributing much more than the others, are often characterized by high abundance, susceptibility to infection / exposure, number of propagules produced per infected individual (Marm [START_REF] Kilpatrick | Host heterogeneity dominates West Nile virus transmission[END_REF][START_REF] Streicker | Differential sources of host species heterogeneity influence the transmission and control of multihost parasites[END_REF][START_REF] Manzoli | Specialist by preference, generalist by need: availability of quality hosts drives parasite choice in a natural multihost-parasite system[END_REF]. For the management of infectious diseases, the way of how parasites and pathogens use their multiple hosts is still challenging [START_REF] Fenton | Are all hosts created equal? Partitioning host species contributions to parasite persistence in multihost communities[END_REF], particularly for epidemiology [START_REF] Carrau | Epidemiological approach to nematode polyparasitism occurring in a sympatric wild ruminant multi-host scenario[END_REF]. With the advent of molecular taxonomy, the cryptic diversity discovered in many animal and plants species revolutionized the study of interactions between species in general, and more particularly host-parasite relationships (Pérez-Ponce de [START_REF] De León | What we don't recognize can hurt us: a plea for awareness about cryptic species[END_REF][START_REF] Pinacho-Pinacho | A hyper-diverse genus of acanthocephalans revealed by tree-based and non-tree-based species delimitation methods: ten cryptic species of Neoechinorhynchus in Middle American freshwater fishes[END_REF]. Indeed, cryptic diversity was found in most parasite groups and many of these parasites, initially considered as generalists, have a narrower host spectrum at the light of the recently revealed genetic divergence [START_REF] Wang | Does multiple hosts mean multiple parasites? Population genetic structure of Schistosoma japonicum between definitive host species[END_REF][START_REF] Janzen | Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity[END_REF][START_REF] Mlynarek | Relative geographic range of sibling species of host damselflies does not reliably predict differential parasitism by water mites[END_REF][START_REF] Escalante | Patterns of cryptic host specificity in duck lice based on molecular data[END_REF][START_REF] De León | An updated look at the uneven distribution of cryptic diversity among parasitic helminths[END_REF][START_REF] Zittel | Cryptic species and their utilization of indigenous and non-indigenous intermediate hosts in the acanthocephalan Polymorphus minutus sensu lato (Polymorphidae)[END_REF]. The reverse is true from a host perspective. Here we will focus on the effect of cryptic diversity on host-parasite relationships in European gammarids. These freshwater amphipods are now a model for studying cryptic diversity. Gammarus fossarum, G. balcanicus and G. roeselii, in particular, showed high numbers of cryptic lineages that are genetically divergent enough to be considered as different species while morphologically homogenous [START_REF] Müller | Mitochondrial DNA variation and the evolutionary history of cryptic Gammarus fossarum types[END_REF][START_REF] Lagrue | Confrontation of cryptic diversity and mate discrimination within Gammarus pulex and Gammarus fossarum species complexes[END_REF][START_REF] Mamos | The legacy of a vanished sea: a high level of diversification within a European freshwater amphipod species complex driven by 15 My of Paratethys regression[END_REF][START_REF] Grabowski | Neogene paleogeography provides context for understanding the origin and spatial distribution of cryptic diversity in a widespread Balkan freshwater amphipod[END_REF][START_REF] Copilaş-Ciocianu | The late blooming amphipods: global change promoted post-Jurassic ecological radiation despite Palaeozoic origin[END_REF][START_REF] Wattier | Continentalscale patterns of hyper-cryptic diversity within the freshwater model taxon Gammarus fossarum (Crustacea, Amphipoda)[END_REF]. Parasite infection and distribution patterns have been studied at the light of these high cryptic divergences, e.g. microsporidia [START_REF] Quiles | Microsporidian infections in the species complex Gammarus roeselii (Amphipoda) over its geographical range: evidence for both host-parasite co-diversification and recent host shifts[END_REF][START_REF] Quiles | Dictyocoela microsporidia diversity and co-diversification with their host, a gammarid species complex (Crustacea, Amphipoda) with an old history of divergence and high endemic diversity[END_REF][START_REF] Quiles | Wide geographic distribution of overlooked parasites: rare Microsporidia in Gammarus balcanicus, a species complex with a high rate of endemism[END_REF], or acanthocephalans [START_REF] Westram | Are cryptic host species also cryptic to parasites? Host specificity and geographical distribution of acanthocephalan parasites infecting freshwater Gammarus[END_REF][START_REF] Galipaud | Variations in infection levels and parasite-induced mortality among sympatric cryptic lineages of native amphipods and a congeneric invasive species: are native hosts always losing?[END_REF]. [START_REF] Westram | Are cryptic host species also cryptic to parasites? Host specificity and geographical distribution of acanthocephalan parasites infecting freshwater Gammarus[END_REF], based on a field study in Swiss rivers, showed that two cryptic lineages of G. fossarum (Gf type A and Gf type B, lineages defined by [START_REF] Müller | Mitochondrial DNA variation and the evolutionary history of cryptic Gammarus fossarum types[END_REF] can be infected by several species of acanthocephalans, including Pomphorhynchus tereticollis and Polymorphus minutus, with higher prevalence in Gf type B. [START_REF] Galipaud | Variations in infection levels and parasite-induced mortality among sympatric cryptic lineages of native amphipods and a congeneric invasive species: are native hosts always losing?[END_REF] showed in a field survey in Eastern France that infection levels by P. tereticollis in G. roeselii are lower than in two other G. pulex/fossarum lineages, but stronger for Pomphorhynchus laevis. They also found highly variable prevalence among G. fossarum lineages. The lineage Gf-III (defined by [START_REF] Lagrue | Confrontation of cryptic diversity and mate discrimination within Gammarus pulex and Gammarus fossarum species complexes[END_REF] showed higher prevalence of P. tereticollis than all other lineages and Gf-II a higher prevalence of P. laevis. The Gf-VII host lineage was poorly infected, suggesting resistance to acanthocephalans for this cryptic species.

These field results suggest some degrees of parasite specificity among G. fossarum cryptic lineages. However, the variations in prevalence observed between acanthocephalan species in the field may be due to at least three factors: (i) a difference in susceptibility to infection, thus reflecting specificity as commonly accepted [START_REF] Poulin | Host specificity under molecular and experimental scrutiny[END_REF]; (ii) a difference in parasite virulence between different hosts that can lead to an artificial deficit of some host-parasite combinations in the field and the absence of parasite accumulation in older hosts [START_REF] Rousset | Inference of parasite-induced host mortality from distributions of parasite loads[END_REF]; (iii) a difference in behavioural manipulation leading to a difference in predation rates. Indeed acanthocephalan parasites use gammarids as intermediate hosts, and change their antipredator behaviour in a way that increase the probability of predation, and thus the probability of transmission to the final host [START_REF] Moore | Altered behavior in cockroaches (Periplaneta americana) infected with an archiacanthocephalan, Moniliformis moniliformis[END_REF][START_REF] Moore | Evolutionary patterns of altered behavior and susceptibility in parasitized hosts[END_REF][START_REF] Lagrue | Modification of hosts' behavior by a parasite: field evidence for adaptive manipulation[END_REF][START_REF] Dianne | Protection first then facilitation: a manipulative parasite modulates the vulnerability to predation of its intermediate host according to its own developmental stage[END_REF][START_REF] Jacquin | Non-specific manipulation of gammarid behaviour by P. minutus parasite enhances their predation by definitive bird hosts[END_REF][START_REF] Fayard | Magnitude and direction of parasite-induced phenotypic alterations: a meta-analysis in acanthocephalans[END_REF]. Therefore, as noticed by [START_REF] Poulin | Host specificity under molecular and experimental scrutiny[END_REF] and [START_REF] Poulin | Host manipulation by parasites: a look back before moving forward[END_REF], only experimental infections can allow us to fully disentangle the causes of the differences in prevalence observed in nature, provided the experiments are ecologically relevant. For example, [START_REF] Bauer | Identifying a key host in an acanthocephalan-amphipod system[END_REF] showed with a laboratory infection experiment that, while higher prevalence in P. laevis are observed in the field, G. roeselii is less susceptible to infection by P. laevis than G. fossarum in experimental conditions. The discrepancy between field and the lab may be due to the lowest behavioural manipulation induced in G. roeselii [START_REF] Bauer | Differential influence of Pomphorhynchus laevis (Acanthocephala) on the behaviour of native and invader gammarid species[END_REF], but the mortality induced by the parasite infection (virulence outside predation probability) was not evaluated. [START_REF] Galipaud | Variations in infection levels and parasite-induced mortality among sympatric cryptic lineages of native amphipods and a congeneric invasive species: are native hosts always losing?[END_REF] showed that there was a difference in parasiteinduced mortality (inferred indirectly from the absence of parasite accumulation in older hosts, see [START_REF] Rousset | Inference of parasite-induced host mortality from distributions of parasite loads[END_REF], and that this mortality was variable between host species. This suggests that the parasites express either differential virulence or differential manipulation depending on the hosts they infect, but this field study does not allow disentangling these two causes of mortality.

We propose here to study the respective roles of some of the factors identified above in the interactions within a community of sympatric hosts and parasites. To this end, we experimentally infected several species of gammarids, including the cryptic species complex Gammarus fossarum, with two sympatric acanthocephalan parasites, P. laevis and P. tereticollis (the latter species being used in experimental infection for the first time to our knowledge). Comparisons of the respective successes of experimental infections between the different host lineages exposed to the parasites will make it possible to test whether they are differently susceptible to the parasites. Monitoring of survival during the whole parasite ontogeny will also be carried out. Several costs may reduce the survival of individuals exposed to infection by acanthocephalans. First, the cost of the penetration of acanthors (early parasite larval stage) into the host digestive wall after ingesting the eggs; second the cost of resistance to infection; or third the cost of harbouring a developing parasite that grows from the acanthor to acanthella to cystacanth stages, multiplying its size by at least a factor of 10. The study of the survival of infected gammarids in the laboratory will allow assessing the virulence of the parasites during their development.

Since no preferential host of P. tereticollis (such as Barbus barbus; [START_REF] Perrot-Minnot | Differential patterns of definitive host use by two fish acanthocephalans occurring in sympatry: Pomphorhynchus laevis and Pomphorhynchus tereticollis[END_REF] was collected during this campaign, the parasites of this species used in this experiment were also collected from the sampled chubs. Chubs are preferential hosts for Pomphorhynchus laevis but can also be infected by P. tereticollis [START_REF] Perrot-Minnot | Differential patterns of definitive host use by two fish acanthocephalans occurring in sympatry: Pomphorhynchus laevis and Pomphorhynchus tereticollis[END_REF]. It should be noted that this infestation by P. tereticollis is, to our knowledge, the first experimental laboratory infection attempt with this species.

The eggs (more precisely acanthor-stage larvae enclosed in a spindle-shaped envelope, cf. [START_REF] Crompton | Biology of the Acanthocephala[END_REF] were extracted from the parasite body by dissection and stored in 2 ml microtubes filled with lab water. Parasite egg suspensions were checked by microscope. We used only batches containing at least 50 % mature eggs (where developed acanthors were clearly visible) in high density. The bodies of the parasites from which the eggs were obtained were preserved in microtubes containing absolute ethanol for genotyping (see genotyping paragraph below). Samples that failed molecular identification were not selected for further experimentation.

Exposure procedure

For each of the parasite species, a suspension was prepared by mixing 7 clutches diluted in water so as to obtain a concentration of 80 eggs per microliter, counted under an optical microscope (Nikon E600 x20). Mixing the clutches of several acanthocephalan females overcomes the possible weakness of a particular female clutch (Franceschi et al. 2010a), and is representative of what may be encountered by gammarids in the natural environment.

Two infestation sessions were carried out one week apart, one using P. laevis parasites, the other with P. tereticollis. Gammarids were starved during the 72 hours preceding the exposure. The exposure procedure described in [START_REF] Franceschi | The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation[END_REF] was then followed. The individuals were placed in pairs in a 60 ml crystallizer (in pairs, the gammarids ingest the leaves better than when they are isolated, unpublished data), in the presence of a piece of elm leaf of about 1 cm² on which a volume of suspension containing an average of 300 parasite eggs were deposited. This dose, higher than those used in our previous experiments, was a compromise between guaranteeing the success of the infection and avoiding a too high rate of multiple infections [START_REF] Franceschi | The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation[END_REF], especially in experimental infections involving co-evolved partners [START_REF] Franceschi | Variation between populations and local adaptation in acanthocephalan-induced parasite manipulation: variation in parasite-induced behavioral manipulation[END_REF][START_REF] Bauer | Identifying a key host in an acanthocephalan-amphipod system[END_REF]. Pieces of leaves without eggs were distributed to the control batches. In each session, some G. pulex from Suzon were also exposed and therefore served as 'positive controls' of infection. After 48 hours, all individuals were individualized in a crystallizer (60 ml) containing lab water and a piece of healthy elm leaf.

Gammarids maintenance and mortality survey

Throughout the duration of the experiment, the crystallizers were inspected daily, the water topped up and a piece of elm leaf added when the former was eaten. Dead gammarids were measured (height of the 4th metacoxal pereion plate) and dissected to check their parasitic status. Dissected tissues were stored in absolute ethanol pending genotyping. Daily monitoring made it possible to amplify the DNA of dead individuals before decay. Every two weeks, one Chironomus plumosus larva (Europrix brand frozen fish food cubes) was distributed in each crystallizer as a dietary supplement, improving survival in the laboratory [START_REF] Labaude | Influence of host nutritional condition on postinfection traits in the association between the manipulative acanthocephalan Pomphorhynchus laevis and the amphipod Gammarus pulex[END_REF], and 24 hours later the water was completely replaced.

Based on the P. laevis development time observed in our previous experiments (e.g. [START_REF] Franceschi | The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation[END_REF]; [START_REF] Labaude | Increased temperature has no consequence for behavioral manipulation despite effects on both partners in the interaction between a crustacean host and a manipulative parasite[END_REF], the gammarids were inspected under a binocular magnifier from 6 weeks post-exposure (Nikon SMZ 745) to check their parasitic status through the cuticle of their host. Individuals detected as infected were isolated. The growth of the parasites was then inspected twice a week. However, some parasites were visible before these 6 weeks (some of them few days after the beginning of the experiment, a development time much too short to correspond to experimental exposure, see results), corresponding to P. tereticollis natural infections (confirmed by genotyping). These animals were analyzed separately.

We ended the experiment when no new developing parasites were detected for 15 successive days. The surviving gammarids were euthanized, measured and dissected to confirm their parasitic status. The developmental stage of the parasites was noted for infected individuals. These gammarids, as well as their parasites, were stored individually in absolute ethanol pending genotyping. The parasites at the acanthella stage had to be genotyped to confirm their species, while the parasites having reached the cystacanth stage were easily recognizable using a binocular magnifier [START_REF] Perrot-Minnot | Larval morphology, genetic divergence, and contrasting levels of host manipulation between forms of Pomphorhynchus laevis (Acanthocephala)[END_REF].

Gammarids & parasites genotyping

To genotype living gammarids, between two and four locomotor appendages were detached and placed in a tube filled with ethanol. For individuals who died during the tracking, as DNA degrades quite quickly after death, a larger amount of tissue was used (between one and three body segments). To genotype adult parasites, a piece of about two mm 3 of body tissue was used to extract DNA. For the parasites at the acanthella stage, the whole individual was used.

All DNA extractions were performed using an extraction kit (EZ-10 96 Well Plate Genomic DNA Isolation kit, BioBasic Inc.) following the manufacturer instructions. Elutions were made in 100 µl or 60 μl of elution buffer provided in the kits, for the gammarids and the parasites, respectively.

For both parasites at the acanthella stage and amphipods, the gene encoding the first subunit of cytochrome oxidase (co1) was amplified using the universal primers LCO1490 and HCO2198 [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF]. To 5 µL of the solution containing the DNA were added 200 nM of each primer, 200 µM of dNTPs, 0.25 U of DNA polymerase (HotStarTaq, Qiagen Inc., Düsseldorf, Germany), 1X of buffer and supplemented with ultrapure water to obtain a total reaction volume of 20 µL. The following thermal treatment was applied: activation of the polymerase for 3 min at 95°C, then 39 cycles: denaturation for 20 s at 95°C, hybridization for 45 s at 40°C and synthesis for 1 min at 65°C, then finally 2 min at 65°C (final synthesis). Amplification success was checked by electrophoresis on 2% agarose gel and visualized with ethidium bromide stain. In case of failure of the co1 amplification for parasites, the ITS region was amplified. The primers BD1f: 5'-GTC GTA ACA AGG TTT CCG TA-3' and ACITS1r: 5'-TTG CGA GCC AAG TGA TTC AC-3' were used [START_REF] Franceschi | The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation[END_REF]. The reaction mixture was subjected to an initial denaturation at 95°C for 3 min, then 39 cycles at 95°C (20 s), 50°C (45 s) and 65°C (45 s). Final elongation was performed for 5 minutes at 65°C.

Adult parasite species were identified by using a RFLP procedure. We first produced our own gene sequences by Sanger sequencing by Genewiz (Leipzig, Germany). The sequences were aligned using Mega software (V10; www.megasoftware.net) and compared to reference sequences (see Supplementary table 1). We then used the Sequence Manipulation Suite (SMS) software (http://www.bioinformatics.org /sms2/rest_map.html) to determine that the restriction enzyme VspI (Thermo Scientific™) enables distinguishing between P. laevis (two restriction fragments of 60 and 620 bp) and P. tereticollis (two fragments of 450 and 240 bp). 10 U of enzyme were added to 1 µL of associated buffer and 5 µl of PCR product and adjusted with ultrapure water to a reaction volume of 15 µL. The mixture was then placed at 37°C for 16 hours and then at 65°C for 20 minutes to inactivate the enzyme. The migration of 15 µL of RFLP product on a 2% agarose gel (for 20 min at 100 V), then visualization with ethidium bromide made it possible to determine the species of the selected samples. For all parasite acanthellae obtained during the survey, PCR products were sequenced and compared to vouchers (see paragraph below for methods).

CO1 amplicons of most gammarid hosts were sequenced and compared to reference sequences (supplementary table 1), using pairwise distances corrected by the Kimura two-parameters (K2P) model (see [START_REF] Lagrue | Confrontation of cryptic diversity and mate discrimination within Gammarus pulex and Gammarus fossarum species complexes[END_REF] and [START_REF] Wattier | Continentalscale patterns of hyper-cryptic diversity within the freshwater model taxon Gammarus fossarum (Crustacea, Amphipoda)[END_REF] for details).

For logistic reasons, 108 gammarids were also typed by using a RFLP procedure developed from the analysis of these sequences using Mega and SMS softwares. A first reaction was carried out to distinguish between the G. fossarum and G. pulex groups: the VspI enzyme (Thermo Scientific™) cuts the DNA of G. pulex into two strands of approximately 200 and 450 base pairs. The G. fossarum amplicons were then subjected to a second RFLP with the DraI enzyme (Thermo Scientific™), to discriminate between the two groups Gf 1-2-3 and Gf 6-7 (species groups identified by [START_REF] Lagrue | Confrontation of cryptic diversity and mate discrimination within Gammarus pulex and Gammarus fossarum species complexes[END_REF]. The PCR products of Gf 6 and Gf 7 are cut into two strands of approximately 450 and 200 base pairs, while the enzyme does not cut the PCR products of Gf 1-2-3. The restriction protocol was the same as detailed before.

All gammarid sequences were submitted to BOLD, in the 'GAMEI' project (https://v4.boldsystems.org/ index.php/MAS_Management_DataConsole?codes=GAMEI).

Statistical analyses

The parasite prevalence were compared by Chi² tests, or by Fisher's exact tests, or using logistic regression with Firth correction in cases of rare events [START_REF] Firth | Bias reduction of maximum likelihood estimates[END_REF]. We have considered as 'infected' all the hosts where a parasite has been detected, whatever its developmental stage.

Host survival was analyzed with R using the survival package [START_REF] Therneau | Modeling survival data: extending the Cox model[END_REF]. Data were censored if animal was still alive at the end of the experiments (118 days for the individuals exposed to P. laevis, and 138 for those exposed to P. tereticollis). Cox models were constructed for comparisons. A preliminary analysis has shown that the survival data did not respect, over the entire follow-up, the condition of proportionality of hazards (checked with the cox.zph function from the survival package) necessary for the application of a Cox regression model. Indeed, an acceleration of mortality is visible after the cystacanth stage was reached (see figures 6 and 8). In addition, during the first weeks of the survey, developing acanthellae were too small to be detected with a binocular magnifier when dissecting dead gammarids, which does not make it possible to distinguish parasitized individuals from uninfected ones. Because of these two constraints, survival has been analyzed in two steps. In the first step, we distinguished only two groups: the control individuals vs. those exposed to the infection. This analysis was stopped at the date where the first experimental acanthella was detected after the dissection of a dead gammarid (44 days for P. laevis and 61 days for P. tereticollis). In the second step, between the date of first detection of acanthella and the end of monitoring, the 'uninfected' and 'parasitized' groups were distinguished among the exposed gammarids. For each analysis, the condition of proportionality of risks was successfully controlled. Finally, to compare the parasites virulence in actually infected gammarids, survival data of infected amphipods were analyzed after the first detection date of P. laevis. Three factors were included in the Cox models: the parasite species, host strain and the intensity of infection (for which data were categorized as follows: one, two, and more than two parasites, following [START_REF] Franceschi | The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation[END_REF]. All possible models with first order interactions were compared by Akaike information criteria, using the dredge function of the MuMin R package (v. 1.43.17 ;[START_REF] Bartoń | MuMIn : multi-model inference[END_REF], and the models minimizing the AICc were presented.

Results

Apart from Gammarus roeselii, which were not taken into account in this experiment (see [START_REF] Bauer | Identifying a key host in an acanthocephalan-amphipod system[END_REF] for their analysis), four host lineages have been found in the gammarids of the Albane River (table 1). Very few Gammarus pulex were found despite the large numbers of collected gammarids. A few Gf 7 individuals were detected by sequencing. Since they were genetically very close to the Gf 6 MOTU and belong to the same breeding group [START_REF] Lagrue | Confrontation of cryptic diversity and mate discrimination within Gammarus pulex and Gammarus fossarum species complexes[END_REF], they were referred as Gf 6 in the dataset and the following analyses. The genotypes of all the hosts were obtained only at the end of the experiments, which explains why it was impossible to restore any imbalances in numbers.

Infection rates

Natural infections Some acanthellae were visible 4 days after the exposure date, in exposed animals, but also in a few unexposed animals (Table 1). Since such a short development time from exposure to acanthella stage is not possible, we inferred that these infections were natural infections for which the quarantine was not long enough for evidencing their development. Most of these infections were P. tereticollis parasites (Figure 1). For the two parasite species, the prevalence were higher in Gf 2 than in Gf 6 (Fisher's exact tests; P. laevis: p = 0.014; P. tereticollis: p < 0.001). No natural infection was observed for Gp D, for which sample size was low (Figure 1).

Experimental infections

Control of the infectivity of parasites using gammarids from Val Suzon Even if the success of infection by P. laevis was low for gammarids from Albane (all MOTUs combined), the high prevalence found in gammarids from the Suzon River showed that the parasites had actually potential for infection (figure 2). In Suzon gammarids, a high P. laevis load was also observed, with an average load of 4.5 parasites per infected hosts (up to 15 parasites in a single individual).

Only 4 individuals from Suzon developed a P. tereticollis infection (two with a single parasite and the other with three). The prevalence of P. tereticollis was similar among gammarids from the two rivers (χ² = 0.001, 1 df, p = 0.970). Infection success in gammarids of the Albane River: prevalence and intensity A variation in the infection rates was observed between host lineages for the two parasite species (Fisher's exact test across all lines for P. laevis, p = 0.018; for P. tereticollis, p < 0.01). Gf 2 was clearly more sensitive than Gf 6 to P. tereticollis (Pearson χ² = 18.105, 1 df, p < 0.001), but was not significantly more sensitive to P. laevis (Pearson χ² = 0.389, 1 df, p = 0.533).

Despite the low sample size, Gp D was found significantly more susceptible than both G. fossarum lineages to infection by P. laevis (Fisher's exact test between Gf 2 and Gp D: p = 0.028; Gf 6 and Gp D: p = 0.016), whereas no experimental infection by P. tereticollis was detected. The average load of P. laevis in Albane gammarids was lower than in those of the Suzon river (Wilcoxon test, W = 1347.5, p < 0.01). Among the Albane gammarids, the average parasite intensity was 2.07 parasites in Gf 2, half of the individuals being mono-infected, and the other half hosting between 2 and 7 parasites. The average intensity was 4 parasites in Gf 6 (six were mono-infected, one harbored 3 parasites, but two individuals presented a very high load of 12 and 15 parasites, which skews the distribution). This difference between Gf 2 and Gf 6 was not significant (Fisher's exact test, p = 0.254). The only two infected Gp D hosted 1 and 2 parasites respectively. Albane's Gf 2 and Gf 6 were mostly mono-infected by P. tereticollis and the maximum load was 3 (the difference in parasite intensity was not significant: Fisher's exact test, p = 1 ; the average intensities being 1.28 for Gf 2 and 1.22 for Gf 6).

Development duration of parasites

The dissections of gammarids that died during the tracking allowed detecting the first experimental P. laevis acanthellae 44 days post-exposure and those of P. tereticollis only after 61 days. The first P. laevis reached the cystacanth stage at 72 days and the first P. tereticollis at 92 days.

We did not observe any difference in P. laevis development duration between Albane and Suzon gammarids (t test, t = 0, p = 1). Since we observed only 3 Suzon gammarids in which developing P. tereticollis reached the cystacanth stage, we could not compare the duration between populations for this parasite. No correlation between the number of parasites and their development duration was observed (P. laevis: Kendall's rank correlation, tau = 0.137, z = 1.210, p = 0.226; P. tereticollis: tau = -0.010, z = -0.0609, p = 0.951). We neither found a difference between development duration according to host lineage, at least between Gf 2 and Gf 6 (Wilcoxon tests, P. laevis: W = 4.5, p = 0.554; P. tereticollis: W = 54, p = 0.809). Therefore, to estimate the parasites development duration to the cystacanth stage, we cumulated data from all infected individuals, whatever their intensity of infection, to maximize sample size. It took significantly less time for P. laevis to reach the cystacanth stage (81 days on average) than for P. tereticollis (106 days) (Mann-Whitney U test: W = 0, p < 0.001), and the variation between early and late cystacanths differentiation was found to be larger in this latter species (figure 4).

Probability of natural and experimental coinfections

Natural infections by P. laevis did not influence significantly subsequent experimental infection, whether by P. laevis (2.63 % probability of infection when not firstly infected (24/912) and 25.0 % in gammarids already infected (1/4), Logistic regression with Firth correction, χ 2 = 2.853, p = 0.091) nor by P. tereticollis (5.34 % (36/674) and 2.77 % (1/3) respectively, Logistic regression with Firth correction: χ 2 = 2.216, p = 0.136), but since numbers were very low for some categories, the relevance of the statistics are questionable.

Natural infection by P. tereticollis strongly influenced an experimental coinfection by P. laevis: while the probability of P. laevis lab infection in gammarids not previously infected was 1.92 % (17/885), it reached 25.81 % (8/31) in gammarids already naturally infected by P. tereticollis (Logistic regression with Firth correction: χ 2 = 25.899, p < 0.0001). For lab infections by P. tereticollis, the previous effect of P. tereticollis natural infection was non-significant (Logistic regression with Firth correction: χ 2 = 0.00, p = 1), the prevalence reaching 5.52% (37/633) in animals uninfected naturally and 0.0% (0/7) in animals already infected.

Parasite virulence

P. laevis-exposed individuals

The results for the Gp D host lineage are not presented because the numbers are too low to be analyzed. In the first part of the tracking, Gf 2 gammarids survive slightly better than Gf 6, and the exposed individuals survived better than the control gammarids (figure 5, table 2). before the first acanthella detection date. In the second part of the tracking, increased mortality of parasitized individuals appeared as soon as the first acanthellae were detected by dissection in Gf 6, but it occurred later, from the date of differentiation of the first cystacanths, in Gf 2 (figure 6, table 2). The survival of uninfected individuals -'eni' and 'controls' -were similar. Globally, survival was better in Gf 2 than in Gf 6.

P. tereticollis-exposed individuals

High mortality of Albane Gammarus exposed to P. tereticollis was observed at the beginning of the survey (compared to P. laevis exposed ones, see Figure 5 and figure 7), but it did not seem to be linked with the confrontation to parasite eggs, since the same trend was observed in both exposed and control gammarids (figure 7, table 3). No significant overall difference in survival was observed between Gf 2 and Gf 6 MOTUs. However, when testing only Gf 6 individuals, exposed animals survived better than control ones (ß = -0.575, z = -2.296, p = 0.022). In the second part of the follow-up and for both MOTUs, parasitized individuals showed increased mortality compared to uninfected, whether these latter are exposed but not infected or controls (figure 8,table 3).

Survival in infected gammarids

Survival analysis in infected gammarids began after the date acanthellae became visible. Interactions were not significant and therefore removed from the presented model (see Supplementary table 2 for the detailed comparison of possible models). We found only a significant effect of parasite species (whole model: χ 2 = 10.69, 3 df, p = 0.01; table 4). Globally, P. laevis-infected gammarids died nearly twice than those infected by P. tereticollis (figure 9a). The observed difference in survival between infected Gf 2 and Gf 6 (whatever the parasite species, figure 9b) was not significant, nor the number of hosted parasites (figure 9c).

Discussion

Susceptibility to infections by different parasite species in different host lineages

We investigated by experimental infections two important factors explaining natural patterns of acanthocephalans infection in the wild: infection success and virulence. Behavioural manipulation, an important factor also explaining differences in prevalence in the field, has not be investigated in this study because of the globally low success of infection. It will be necessary to further evaluate differences in manipulation among cryptic hosts to get a complete prospect of the link between host genotype and efficient exploitation by Pomphorhynchus spp. acanthocephalans.

To our knowledge, this work presents the first results of an experimental infestation of Gammarus spp. with Pomphorhynchus tereticollis. The experimental infestations with P. tereticollis led to a relatively high infection rate in Gf 2 MOTU of Gammarus fossarum, and to a lesser extent in Gf 6 and Gammarus pulex. They did not generate any infection in the few Gp D from the Albane River, but produced a few in G. pulex from Val-Suzon River. High prevalence of P. tereticollis in G. fossarum has been already found in previous field works [START_REF] Westram | Are cryptic host species also cryptic to parasites? Host specificity and geographical distribution of acanthocephalan parasites infecting freshwater Gammarus[END_REF][START_REF] Galipaud | Variations in infection levels and parasite-induced mortality among sympatric cryptic lineages of native amphipods and a congeneric invasive species: are native hosts always losing?[END_REF][START_REF] Harris | The life cycle of the parasite Pomphorhynchus tereticollis in reference to 0+ cyprinids and the intermediate host Gammarus spp in the UK[END_REF]. Our observation that the G. pulex from Val-Suzon are not particularly susceptible to P. tereticollis (in any case much less than to P. laevis) contradicts the hypothesis proposed by [START_REF] Franceschi | Variation between populations and local adaptation in acanthocephalan-induced parasite manipulation: variation in parasite-induced behavioral manipulation[END_REF] which stated that amphipods of this site are more sensitive to infections by Pomphorhynchus parasites because this population is naïve to these parasites. [START_REF] Harris | The life cycle of the parasite Pomphorhynchus tereticollis in reference to 0+ cyprinids and the intermediate host Gammarus spp in the UK[END_REF] found similar results in England about P. tereticollis infections: high prevalences were found in G. fossarum (up to 90 %), but no parasites (cystacanth stages) in G. pulex. We could therefore propose a general lower susceptibility of G. pulex to P. tereticollis compared to G. fossarum, but that the MOTU of G. fossarum matters. Our finding that Gf 2 is more sensitive to infection than Gf 6 sharply contrasts with field data from [START_REF] Galipaud | Variations in infection levels and parasite-induced mortality among sympatric cryptic lineages of native amphipods and a congeneric invasive species: are native hosts always losing?[END_REF] where the prevalence in P. tereticollis was found similar between these MOTUs, and highlights the usefulness of experimental infections in deciphering host-parasite relationships [START_REF] Poulin | Host manipulation by parasites: a look back before moving forward[END_REF]. This is particularly true for acanthocephalans, where the prevalence in natura reflects a combination of several processes due to the long intra-host development time [START_REF] Bauer | Identifying a key host in an acanthocephalan-amphipod system[END_REF]. One of these processes is parasite virulence that we will discuss thereafter.

This study confirms, under controlled conditions, that all MOTUs present in the Albane River can be infected by P. laevis. The infection success in Gf 2 and Gf 6 is of the same order of magnitude. Experimental infections tend to show that Gp D seems to be particularly sensitive to this parasite compared to Gf 2 and Gf 6. These results are in contradiction with those of [START_REF] Galipaud | Variations in infection levels and parasite-induced mortality among sympatric cryptic lineages of native amphipods and a congeneric invasive species: are native hosts always losing?[END_REF] who found low in natura prevalence of P. laevis cystacanths in G. pulex. However, as showed by [START_REF] Bauer | Identifying a key host in an acanthocephalan-amphipod system[END_REF], a low prevalence of cystacanths found after direct collection in nature could simply mean that either parasite virulence is high, or that the parasite increased so much the predation rate of its host that parasitized individuals disappear rapidly from populations. Here, our measures of prevalence were made after quarantine or after experimental infection, i.e. in conditions where predation was absent or the survival controlled. In addition, the Suzon population, composed of G. pulex [START_REF] Labaude | Influence of host nutritional condition on postinfection traits in the association between the manipulative acanthocephalan Pomphorhynchus laevis and the amphipod Gammarus pulex[END_REF], is very sensitive to infection, mirroring the few G. pulex from the Albane River. Alternatively, despite our precautions to standardize the infections, we cannot completely rule out an artefact linked to the experimental conditions. Indeed, even if we controlled the dose of parasite eggs provided to the gammarids, we are not able to control how many eggs have actually been consumed. G. pulex is usually larger than G. fossarum [START_REF] Nijssen | Some notes on the distribution and the ecology of the amphipod Gammarus fossarum Koch, 1835, in the Netherlands (Crustacea, Malacostraca)[END_REF], and because food consumption is positively correlated to size in Gammarus [START_REF] Sutcliffe | Effects of diet, body size, age and temperature on growth rates in the amphipod Gammarus pulex[END_REF], this species may have consumed more eggs than the other MOTUs. However, if this hypothesis may possibly explain the differences in infection success obtained for P. laevis, it certainly cannot explain the results of the infestation with P. tereticollis, for which G. pulex appeared to be poorly sensitive. We therefore propose that P. laevis is a parasite better adapted to G. pulex than to G. fossarum (whatever the genetic lineage).

Our results therefore highlight the need for exposing each of the MOTUs of the G. pulex / fossarum complex species to experimental infections to understand their relative sensitivity to the different species of acanthocephalans.

Natural and experimental coinfections

Natural infection by P. tereticollis favours a further interspecific experimental infection by P. laevis. Contrastingly, natural infection by P. tereticollis did not influence intraspecific experimental superinfections by P. tereticollis, and natural infection by P. laevis did not influence neither a secondary infection by another acanthocephalan. However, sample size are sometimes so low that the statistical power of these later analyses are questionable. Cornet et al. (2009) showed that the level of immune defences of G. pulex is strongly depressed by the acanthocephalans P. laevis, P. tereticollis, and that acanthocephalan infection favors further superinfection by Escherichia coli bacteria. However, Dianne et al. (2010), using successive experimental infections by P. laevis, did not observed any increase of acanthocephalan intraspecific superinfection. It is therefore possible that the immune depression only favours interspecific parasite/pathogens superinfections but not intraspecific ones, suggesting that this immune depression is a costly by-product of the acanthocephalan infections. Since multiple infections are the rule in nature, further experimental investigations are welcome, particularly for the study of the evolution of virulence [START_REF] Alizon | Multiple infections and the evolution of virulence[END_REF].

Development duration of parasites

Acanthocephalans development time in their intermediate hosts is strongly influenced by the environment [START_REF] Awachie | The development and life history of Echinorhynchus truttae Schrank, 1788 (Acanthocephala)[END_REF][START_REF] Lackie | The effect of temperature on the development of Moniliformis dubius (Acanthocephala) in the intermediate host, Periplaneta americana[END_REF][START_REF] Tokeson | The effects of temperature and oxygen on the development of Polymorphus marilis (Acanthocephala) in Gammarus lacustris (Amphipoda)[END_REF][START_REF] Brattey | Life history and population biology of larval Acanthocephalus lucii (Acanthocephala: Echinorhynchidae) in the isopod Asellus aquaticus[END_REF][START_REF] Labaude | Increased temperature has no consequence for behavioral manipulation despite effects on both partners in the interaction between a crustacean host and a manipulative parasite[END_REF], highlighting the necessity of studying this life-history trait under controlled conditions. The development duration of Polymorphus minutus and Pomphorhynchus laevis under laboratory conditions have already been studied [START_REF] Hynes | The development of Polymorphus minutus (Goeze, 1782) (Acanthocephala) in the intermediate host[END_REF][START_REF] Butterworth | The development of the body wall of Polymorphus minutus (Acanthocephala) in its intermediate host Gammarus pulex[END_REF][START_REF] Franceschi | The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation[END_REF][START_REF] Labaude | Influence of host nutritional condition on postinfection traits in the association between the manipulative acanthocephalan Pomphorhynchus laevis and the amphipod Gammarus pulex[END_REF][START_REF] Labaude | Increased temperature has no consequence for behavioral manipulation despite effects on both partners in the interaction between a crustacean host and a manipulative parasite[END_REF], but Pomphorhynchus tereticollis development was not documented before this study. The development time in P. laevis observed here was similar to the one found by [START_REF] Labaude | Increased temperature has no consequence for behavioral manipulation despite effects on both partners in the interaction between a crustacean host and a manipulative parasite[END_REF] at a similar temperature. The development of P. tereticollis took 23 % longer than P. laevis under the same conditions. The among-host variation in maturation time (time between the appearance of the first and the last cystacanths) was also larger and more variable than that of P. laevis. This longer development time explains a posteriori why we observed so many natural P. tereticollis infections after the quarantine we imposed to gammarids, since the quarantine time was estimated from our previous experience on P. laevis development.

Virulence

Ingestion of parasites and the early beginning of larval development did not negatively affect the host survival. Therefore, the wounds inflicted when the acanthors pierce the digestive tract of the arthropod to pass into the general cavity, and/or a possible early resistance to infection (cellular response, humoral and immune encapsulation [START_REF] Crompton | Biology of the Acanthocephala[END_REF] were not costly enough to increase mortality. The literature is contradictory about the host survival in early stages of infection by acanthocephalans. Increased mortality shortly after acanthocephalan experimental exposure was observed by [START_REF] Crompton | Biology of the Acanthocephala[END_REF] and [START_REF] Hynes | The development of Polymorphus minutus (Goeze, 1782) (Acanthocephala) in the intermediate host[END_REF], but these studies were probably carried out with massive doses of eggs. This dose was not specified in these studies, but due to the dose-dependant prevalence [START_REF] Franceschi | The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation[END_REF], and due to the high prevalence observed in these studies, this hypothesis is reasonable to consider. In these cases, the damages inflicted by many acanthors crossing the digestive tract wall may have been deleterious. On the other hand, [START_REF] Uznanski | Parasite population regulation: lethal and sublethal effects of Leptorhynchoides thecatus (Acanthocephala: Rhadinorhynchidae) on Hyalella azteca (Amphipoda)[END_REF] did not find any increased mortality in the first 24 hours post-exposure in Hyalella azteca exposed to Leptorhynchoides thecatus. Yet, here again, the high infection success suggested the usage of high doses of eggs. Even more intriguing, experimental infections of Asellus by Acanthocephalus lucii studied by [START_REF] Brattey | Life history and population biology of larval Acanthocephalus lucii (Acanthocephala: Echinorhynchidae) in the isopod Asellus aquaticus[END_REF] and [START_REF] Benesh | Proximate factors affecting the larval life history of Acanthocephalus lucii (Acanthocephala)[END_REF] induced early increased mortalities, whereas [START_REF] Hasu | Costs of parasite resistance for female survival and parental care in a freshwater isopod[END_REF] found a better survival of infected individuals on the same biological models. This later, intriguing, result is what we observed in the present study: gammarids exposed to parasite eggs showed improved survival in the early days post-exposure, i.e. before the infection can be detected. Only speculative discussions on these observations can be proposed because it was not possible to ensure whether individuals dying at the beginning of the survey were infected or not. At least can we exclude a selective mortality due to the exposure. One of the speculative explanation is that the observed improved survival of exposed gammarids could be part of "parasite manipulation", where the parasite would protect its host from mortality, before becoming infective to the next host [START_REF] Parker | When should a trophically transmitted parasite manipulate its host?[END_REF]. Such a protection was described for P. laevis in the late acanthella stage [START_REF] Dianne | Protection first then facilitation: a manipulative parasite modulates the vulnerability to predation of its intermediate host according to its own developmental stage[END_REF][START_REF] Rigaud | Time-course of antipredator behavioral changes induced by the helminth Pomphorhynchus laevis in its intermediate host Gammarus pulex: the switch in manipulation according to parasite developmental stage differs between behaviors[END_REF], but this was a protection against mortality due to predation, which is not the case here. A mechanistic explanation of such a phenomenon would be that the early stages of the parasite divert some energy of the host for improving its survival. Castrating parasites are known to divert the energy normally allocated to host reproduction to their own purpose, in particular for the survival of the host they infect [START_REF] Benesh | Proximate factors affecting the larval life history of Acanthocephalus lucii (Acanthocephala)[END_REF]. Gammarid acanthocephalans are known to castrate their hosts, partially [START_REF] Bollache | Effects of two acanthocephalan parasites on the fecundity and pairing status of female Gammarus pulex (Crustacea: Amphipoda)[END_REF] or totally [START_REF] Bollache | Effects of two acanthocephalan parasites on the fecundity and pairing status of female Gammarus pulex (Crustacea: Amphipoda)[END_REF][START_REF] Bailly | Stage-dependent behavioural changes but early castration induced by the acanthocephalan parasite Polymorphus minutus in its Gammarus pulex intermediate host[END_REF]. It is not known precisely at what stage of parasite growth castration occurs in Pomphorhynchus, but it happens early during Polymorphus ontogeny, at least before the acanthellae are visible in their host [START_REF] Bailly | Stage-dependent behavioural changes but early castration induced by the acanthocephalan parasite Polymorphus minutus in its Gammarus pulex intermediate host[END_REF]. Alternatively, egg parasites eaten by gammarids, but unable to develop, can themselves become a form of diet supplementation for the hosts that 'prey' on them [START_REF] Goedknegt | Parasites as prey[END_REF], improving host survival.

Parasite virulence was expressed in our study when the acanthellae enter their exponential phase of growth. It is interesting to note, even if the differences observed are not always supported statistically, that Gf 6 was more sensitive to Pomphorhynchus infections than Gf 2, whatever the parasite species. In Hyalella azteca infected by Corynosoma constrictum, a higher mortality was observed at the time of rapid development of the parasite larvae, only in heavily infected hosts [START_REF] Duclos | Virulence of Corynosoma constrictum (Acanthocephala: Polymorphidae) in Hyalella azteca (Amphipoda) throughout parasite ontogeny[END_REF]. This increased mortality due to acanthocephalan infections is interpreted as being due to displacement of the host's organs [START_REF] Bentley | Pomphorhynchus laevis (Acanthocephala): elevation of haemolymph protein concentrations in the intermediate host, Gammarus pulex (Crustacea: Amphipoda)[END_REF][START_REF] Dezfuli | Crustacean-acanthocephalan interaction and host cell-mediated immunity: parasite encapsulation and melanization[END_REF], or effects on metabolism [START_REF] Rumpus | The effect of the acanthocephalan Pomphorhynchus laevis upon the respiration of its intermediate host, Gammarus pulex[END_REF]. In our study, the energy requirements of the two parasites are probably different, since P. tereticollis larvae are larger than P. laevis [START_REF] Perrot-Minnot | Larval morphology, genetic divergence, and contrasting levels of host manipulation between forms of Pomphorhynchus laevis (Acanthocephala)[END_REF]. However, the virulence of P. tereticollis was weaker than that of P. laevis. An effect on metabolism would therefore only be possible if one considers that P. laevis grows faster than P. tereticollis, which could exhaust the host's metabolism at higher intensity. If so, this would be in line with the "boom-bust" and "slow and steady" strategies proposed by [START_REF] Clec'h | Striking differences in virulence, transmission and sporocyst growth dynamics between two schistosome populations[END_REF] for schistosome infections. Studies looking at metabolism of infected gammarids have only been carried out at parasite cystacanth stages [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] Korkofigas | Acanthocephalan-related variation in the pattern of energy storage of a behaviorally and physiologically modified host: field data[END_REF]. All these studies showed that parasitized amphipods are deprived of lipid and protein reserves, while they have higher glycogen rates. This shows high demand for immediate energy (glycogen) in infected amphipods, which are forced to draw on their reserves (lipids), probably at the benefit of the parasite metabolism. Such a strategy is common in parasites. In schistosomes, such effects on the host fitness and energy diversion occur both early and in the late development of the parasite [START_REF] Jong-Brink | Multiple strategies of schistosomes to meet their requirements in the intermediate snail host[END_REF]. In Plasmodium, the intra-host competition in cases of multiple infections is leading to increase energetic demand due to host overexploitation by the faster-developing parasites [START_REF] De Roode | Dynamics of multiple infection and within-host competition in genetically diverse malaria infections[END_REF]. Differences in survival between Gf 2 and Gf 6 hosts may be due to differences in metabolism, a hypothesis that necessitate further investigations. Another possible explanation for a differential in survival between host lineages could be differences in the ability of acanthocephalans to reduce the intensity of the immune responses of their hosts (Cornet et al., 2009a). On the one hand, this partial immunosuppression might be advantageous for the host, owing the cost of mounting an immune response, either in terms of physiology [START_REF] Moret | Survival for immunity: the price of immune system activation for Bumblebee workers[END_REF] or because of immune cytotoxicity of enzymes used for the invertebrate immune responses [START_REF] Sadd | Self-harm caused by an insect's innate immunity[END_REF]. On the other hand, as discussed before, this immunosuppression can make hosts more susceptible to other infections, such as bacterial infections (Cornet et al., 2009a;[START_REF] Cornet | Parasite virulence when the infection reduces the host immune response[END_REF]. This immune modification is not observed in all gammarids: infections of various populations of G. roeselii by P. laevis do not show immunosuppression, with some populations even showing an immune response to the infection [START_REF] Moret | Is the host or the parasite the most locally adapted in an amphipod-acanthocephalan relationship? A case study in a biological invasion context[END_REF]. In addition, the level of immune defenses are variable among gammarid populations (Cornet et al., 2009b). Therefore, Gf 2 and Gf 6 could be immunosuppressed differently, a hypothesis that remains to be tested.

General conclusion

Our results complement those of a previous study on Acanthocephala, carried out in natura by counting infections by cystacanths [START_REF] Galipaud | Variations in infection levels and parasite-induced mortality among sympatric cryptic lineages of native amphipods and a congeneric invasive species: are native hosts always losing?[END_REF]. G. pulex might be a more important host than G. fossarum for the persistence of P. laevis in the rivers. However, differences in susceptibility to P. tereticollis were more pronounced between different host genotypes within the G. fossarum species complex than between G. fossarum and G. pulex. Our data therefore confirm that the specificity to a given parasite does not necessarily depend on the phylogenetic distance between the hosts, but indicate that we can no longer ignore cryptic hosts' genetic information to determine the level of specialization of the parasites. In addition to differences in susceptibility to infection, we also showed that a strong virulence is expressed when P. laevis or P. tereticollis reached the cystacanth stage, but that the different genotypes of G. fossarum were not affected with the same intensity.

Figure 1 -

 1 Figure 1 -Infections by P. laevis and P. tereticollis of natural origin in gammarid MOTUs from Albane River. Numbers of infected amphipods are indicated above the bars, and total numbers of each MOTU appear below.

Figure 2 -

 2 Figure 2 -Success of laboratory infections by P. laevis and P. tereticollis, in gammarids (all MOTUs together) coming from the two rivers Albane and Suzon. Numbers of infected gammarids are given above the bars, and total sample sizes from each river below the bars.

Figure 3 -

 3 Figure 3 -Success of experimental infections by the two parasite species according to the host lineages present in the Albane River. Numbers of infected gammarids are given above the bars, and total sample sizes for each MOTU are below the bars.

Figure 4 -

 4 Figure 4 -Development duration of parasites in the two species until the cystacanth stage, in days post-exposure. Thick black bars indicate medians, boxes are the upper and lower quartiles, and whiskers represent the upper and lower values. Numbers are sample size.

Figure 5 -

 5 Figure 5 -Survival of P. laevis exposed gammarid MOTUs (plain lines) and controls (dotted lines),

Figure 6 -

 6 Figure 6 -Survival of P. laevis exposed gammarid MOTUs and controls (dotted lines), after the first acanthella detection date. Infected individuals (plain lines) can now be distinguished from exposed but not infected ones ('eni', in dashed lines). Letters on the right of the graphs indicate the statistical groups. The vertical orange lines denote the first cystacanth detection date.

Figure 7 -

 7 Figure 7 -Survival of P. tereticollis exposed gammarid MOTUs (plain lines) and controls (dotted lines), before the first acanthella detection date.

Figure 8 -

 8 Figure 8 -Survival of P. tereticollis exposed gammarid MOTUs and controls (dotted lines), after the first acanthella detection date. Infected individuals (plain lines) can now be distinguished from exposed but not infected ones ('eni', in dashed lines). Letters on the right of the graphs indicate the statistical groups. The vertical orange lines denote the first cystacanth detection date.

Figure 9 -

 9 Figure 9 -Comparison of survival of infected gammarids after the first P. laevis acanthella detection date: a) effect of parasite species; b) effect of host MOTU; c) effect of the intensity of infection.

Table 1 .

 1 Numbers of gammarid MOTUs found in each exposure session, according to their infection status, including individuals with natural infections in brackets (by parasite species: P. laevis in red, P. tereticollis in green, and undetermined in black ; & for a mixed P. laevis and P. tereticollis

	infection).
			P. laevis exposure
	MOTU	controls early death uninfected	early death	exposed infected	eni	total
	Gf 2	7 (1+1)	18	33	14 (7)	443 (3+23)	515
	Gf 6	12	52	58	9 (1+1)	408	539
	Gp D	1	0	1	2	6	10
	undetermined	1	4	4	0	34	43
	"Suzon"	NA	NA	2	79	18	99
			P. tereticollis exposure
	MOTU	controls		exposed		total
		early death	uninfected	early death	infected	eni
	Gf 2	25 (2)	23	119 (1+11+6)	28 (1)	236 (2+6)	431
	Gf 6	30 (1)	12	147 (1+2)	9	372	570
	Gp D	0	0	19	0	10	29
	undetermined	3	2 (1)	22 (1&1+1)	0	22 (1)	49
	"Suzon"	NA	NA	12	4	81	97

Gf 2 = Gammarus fossarum MOTU 2 ; Gf 6 = Gammarus fossarum MOTU 6 ; Gp D = Gammarus pulex MOTU D. Undetermined: gammarids which could not by genotyped (and therefore removed from statistical analyses). Suzon: Gammarus pulex from Suzon, not distinguished by MOTU. Controls:

gammarids not exposed to parasite eggs. Exposed: gammarids experimentally exposed to parasite eggs. Early death: gammarids that died before the date of detection of the first acanthella. Eni (exposed not infected): exposed gammarids that did not develop any infection.

Table 2 .

 2 Results of the Cox models analysing the survival of gammarids exposed to P. laevis as a function of gammarid lineages and parasite treatment, divided according to the parasite development stage (before the first acanthella detection date [1st part] and after this date [2nd part]). The interaction between factors was not significant, and was removed from the model.

	Global risks proportionality: χ² = 0.821, 2 df, p = 0.66 for the first part; χ² = 1.558, 3 df, p = 0.67 for
	the second part.
	Development	Comparison	ß	odd ratio	± s.e.	z	df	p
	stage
	1st part	MOTU: Gf 2 vs. Gf 6	0.463	1.589	0.204	2.273	1	0.023
		Treatment: exposed vs.	-0.655	0.519	0.267	-2.453	1	0.014
		control
	2nd part	MOTU: Gf 2 vs. Gf 6	0.528	1.695	0.184	2.869	1	0.004
		Treatment: global						<0.001
		-control vs. 'eni'	0.103	1.108	0.349	0.294	1	0.768
		-control vs. infected	-1.912	0.148	0.489	-3.906	1	<0.001
		-'eni' vs. infected	-1.809	0.164	0.368	-4.917	1	<0.001

Table 3 .

 3 Results of the Cox model analysing the survival of gammarids exposed to P. tereticollis as a function of gammarid MOTUs and parasite treatment, divided according to the parasite development stage (before the first acanthella detection date [1st part] and after this date [2nd part]). The interaction between factors was not significant, and was removed from the model.

	Global risks proportionality: χ² = 4.47, 2 df, p = 0.107 for the first part; χ² = 1.717, 3 df, p = 0.63 for
	the second part.
	Development	Comparison	ß	odd ratio	± s.e.	z	df	p
	stage
	1st part	MOTU: Gf 2 vs. Gf 6	-0.017	0.983	0.123	-0.138	1	0.809
		Treatment: exposed vs.	-0.297	0.743	0.192	-1.552	1	0.121
		control
	2nd part	MOTU: Gf 2 vs. Gf 6	0.777	2.175	0.209	3.712	1	<0.001
		Treatment: global						<0.001
		-control vs. 'eni'	-0.383	0.682	0.424	-0.904	1	0.366
		-control vs. infected	0.982	2.669	0.484	2.029	1	0.042
		-'eni' vs. infected	-1.365	0.255	0.288	-4.738	1	<0.001

Table 4 .

 4 Results of the Cox model analysing the survival of infected gammarids as a function of parasite species, gammarid MOTUs and intensity of infection, after the first P. laevis acanthella detection date. The interaction between factors were not significant and removed from the model.

	Global risks proportionality: χ² = 3.085, 3 df, p = 0.38.
	Source	ß	odd ratio	± s.e.	z	df	p
	Parasite species	-0.958	0.384	0.422	-2.268	1	0.023
	MOTU: Gf 2 vs. Gf 6	0.773	2.166	0.416	1.856	1	0.063
	Parasite number: 1, ≥2	0.767	2.125	0.433	1.769	1	0.077

  Supplementary table 2 -Model selection table for the survival analysis of infected gammarids(MuMin R package). Factors compared : intensity of infection (nb), host MOTU (motu), parasite species (para) and their first order interactions.

			Factors						Analysis parameters
				nb	nb	motu
	nb	motu	para	×	×	×	adj R²	df	logLik	AICc delta	weight
			motu	para	para
	+	+	+				0.348	3	-84.9	179.9	0.00	0.163
		+	+				0.267	2	-86.3	177.2	0.31	0.140
			+				0.194	1	-87.5	177.2	0.33	0.139
	+		+				0.256	2	-86.5	177.5	0.69	0.116
	+	+	+		+		0.367	4	-84.5	179.0	2.13	0.056
	+	+					0.212	2	-87.2	179.0	2.15	0.056
		+					0.131	1	-88.4	179.1	2.43	0.049
	+	+	+			+	0.359	4	-86.2	179.6	2.71	0.042
		+	+			+	0.273	3	-84.8	179.7	2.80	0.040
	+	+	+	+			0.349	4	-84.8	179.7	2.80	0.040
	+		+		+		0.265	3	-86.4	179.8	3.00	0.036
							0.000	0	-90.2	180.4	3.54	0.028
	+						0.067	1	-89.3	180.8	3.99	0.022
	+	+	+		+	+	0.389	5	-84.0	181.2	4.39	0.018
	+	+		+			0.212	3	-87.2	181.6	4.74	0.015
	+	+	+	+	+		0.368	5	-84.5	181.1	5.23	0.012
	+	+	+	+		+	0.362	5	-84.6	182.3	5.48	0.011
	+	+	+	+	+	+	0.390	6	-84.0	184.7	7.87	0.003

Acknowledgements

We thank Jean-Emmanuel Rollin for field sampling.

Data, scripts, code, and supplementary information availability

The dataset (in French) and data description file explaining the variables are available on the data.indores.fr data repository.

Funding

This study was funded by the French Agence Nationale de la Recherche (ANR), ANR-13-BSV7-0004-01

Appendices

Supplementary table 1 -Accession numbers of the reference sequences used to assign the gammarid MOTUs and the parasite species

Conflict of interest disclosure

The authors declare that they comply with the PCI rule of having no financial conflicts of interest in relation to the content of the article.

TR is recommender for PCI infections.