Annia Alba
email: annia.alba@gmail.com

Guillaume Tetreau

Cristian Chaparro

Jorge Sánchez

Antonio A Vázquez

Benjamin Gourbal
email: benjamin.gourbal@univ-perp.fr

Natural resistance to Fasciola hepatica (Trematoda) in Pseudosuccinea columella snails: A review from literature and insights from comparative "omic" analyses

Keywords: Transcriptome -Albumen gland -Allocation of resources -Response to stress -Immune defense -Cost of resistance Bge cells, Biomphalaria glabrata embryonic cell line, G-CSFR, granulocyte colony stimulatory factor, IRF, interferon regulatory factors, LBP/BPI, lipopolysaccharide-binding protein/bactericidal permeability

The snail Pseudosuccinea columella is one of the main vectors of the medically-important trematode Fasciola hepatica. In Cuba, the existence of natural P. columella populations that are either susceptible or resistant to F. hepatica infection offers a unique snail-parasite for study of parasite-host compatibility and immune function in gastropods. Here, we review all previous literature on this system and present new "omic" data that provide a molecular baseline of both P. columella phenotypes from naïve snails. Comparison of whole snail transcriptomes (RNAseq) and the proteomes of the albumen gland (2D-electrophoresis, MS) revealed that resistant and susceptible strains differed mainly in an enrichment of particular biological processes/functions and a greater abundance of proteins/transcripts associated with immune defense/stress response in resistant snails. These results indicate a differential allocation of molecular resources to self-maintenance and survival in resistant P. columella that may cause enhanced responsiveness to stressors (i.e. F. hepatica infection or tolerance to variations in environmental pH/total water hardness), possibly as trade-off against reproduction and the ecological cost of resistance previously suggested in resistant populations of P. columella

Introduction

Among digenean parasites that are transmitted by snail vectors, those causing fascioliasis (Fasciola spp.) and mainly Fasciola hepatica have gained particular interest due to their wide distribution, their impact on veterinary health and on the economy, in association with livestock infections [START_REF] Khan | The global burden of fasciolosis in domestic animals with an outlook on the contribution of new approaches for diagnosis and control[END_REF][START_REF] Mehmood | A review on epidemiology, global prevalence and economical losses of fasciolosis in ruminants[END_REF], and their re-emergence as human pathogens [START_REF] Mas-Coma | Chapter 2. Fasciola, lymnaeids and human fascioliasis, with a global overview on disease transmission, epidemiology, evolutionary genetics, molecular epidemiology and control[END_REF]. Fasciola spp. have a two-host life cycle (i.e. mammals and snails as definitive and intermediate hosts, respectively), and transmission in a specific geographical area is mostly dependent on presence of vector snails of the family Lymnaeidae. In this sense, host efficiency may be influenced by the local adaptation between parasite and snail species [START_REF] Dar | Variations in local adaptation of allopatric Fasciola hepatica to French Galba truncatula in relation to parasite origin[END_REF][START_REF] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF].

Worldwide, 30 lymnaeid species are known as intermediate hosts of Fasciola spp. (Vázquez et al., 2018). Particularly, Pseudosuccinea columella (formerly known as Lymnaea columella), considered one of the main intermediate host species for transmission of F. hepatica, is an efficient invasive snail species that has attained wide geographical distribution [START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at worldwide scale[END_REF][START_REF] Vázquez | Lymnaeid snails hosts of Fasciola hepatica and Fasciola gigantica (Trematoda: Digenea): a worldwide review[END_REF]. Presumed to have originated in North America, the high adaptability and invasive capacity of few genotypes have allowed establishment of P. columella in several parts of the world (for details, see [START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at worldwide scale[END_REF]. Nowadays, P. columella is reported in South America and the Caribbean [START_REF] Cucher | PCR diagnosis of Fasciola hepatica in field-collected Lymnaea columella and Lymnaea viatrix snails[END_REF][START_REF] Gutiérrez | First report of larval stages of Fasciola hepatica in a wild population of Pseudosuccinea columella from Cuba and the Caribbean[END_REF], as well as in Africa [START_REF] Brown | Freshwater Snails of Africa and Their Medical Importance[END_REF], Australia [START_REF] Molloy | The distribution of Fasciola hepatica in Queensland, Australia, and the potential impact of introduced snail intermediate hosts[END_REF], several Pacific islands [START_REF] Cowie | Invertebrate invasions on Pacific Islands and the replacement of unique native faunas: a synthesis of the land and freshwater snails[END_REF][START_REF] Pointier | Taxonomy and distribution of freshwater mollusks of French Polynesia[END_REF] and Europe [START_REF] Pointier | Pseudosuccinea columella (Say 1817) (Gastropoda, Lymnaeidae), snail host of Fasciola hepatica: first record for France in the wild[END_REF].

Concomitantly, the global distribution of this snail species has also complicated the epidemiological scenario of fascioliasis [START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at worldwide scale[END_REF]. Transmission of F. hepatica by P. columella is well documented in and outside of its native range (e.g. [START_REF] Gutiérrez | First report of larval stages of Fasciola hepatica in a wild population of Pseudosuccinea columella from Cuba and the Caribbean[END_REF][START_REF] Cruz-Reyes | Suitability of six lymnaeid snails for infection with Fasciola hepatica[END_REF][START_REF] Cucher | PCR diagnosis of Fasciola hepatica in field-collected Lymnaea columella and Lymnaea viatrix snails[END_REF] and P. columella snails have been found also naturally infected with Fasciola gigantica [START_REF] Grabner | Invasion biology meets parasitology: a case study of parasite spill-back with Egyptian Fasciola gigantica in the invasive snail Pseudosuccinea columella[END_REF]. Fasciola hepatica infection in this species does reduce snail fecundity [START_REF] Gutiérrez | Fasciola hepatica (Trematoda: Digenea): its effect on the life history traits of Pseudosuccinea columella (Gastropoda: Lymnaeidae), an uncommon interaction[END_REF][START_REF] Salazar | Effect of the exposure to Fasciola hepatica (Trematoda: Digenea) on life history traits of Lymnaea cousini and Lymnaea columella (Gastropoda: Lymnaeidae)[END_REF], but is also characterized by higher redial and metacercariae productivity compared to Galba truncatula [START_REF] Dar | Fasciola hepatica: development of redial generations in experimental infections of Pseudosuccinea columella[END_REF][START_REF] Vignoles | Fasciola hepatica: comparative metacercarial productions in experimentally-infected Galba truncatula and Pseudosuccinea columella[END_REF] and greater survival when compared to species of Galba [START_REF] Salazar | Effect of the exposure to Fasciola hepatica (Trematoda: Digenea) on life history traits of Lymnaea cousini and Lymnaea columella (Gastropoda: Lymnaeidae)[END_REF][START_REF] Vignoles | Fasciola hepatica: comparative metacercarial productions in experimentally-infected Galba truncatula and Pseudosuccinea columella[END_REF].

Lymnaeid snails and F. hepatica transmission in Cuba: resistant and susceptible populations of P. columella

In Cuba, F. hepatica is transmitted by two species of snails: Galba cubensis, considered the main intermediate host (natural prevalence ranged from 1 to 34%; [START_REF] Alba | Assessment of the FasciMol-ELISA in the detection of the trematode Fasciola hepatica in field-collected Galba cubensis: a novel tool for the malacological survey of fasciolosis transmission[END_REF]Vázquez et al., 2015), and P. columella (Alba et al. under review, Gutiérrez et al., 2011). The latter has a more discrete distribution than G. cubensis [START_REF] Vázquez | Distribución y preferencia de hábitats de moluscos hospederos intermediarios de Fasciola hepatica en Cuba[END_REF]; to date reported from only 68 localities in western and central Cuba with no records in the easternmost region of the island (Alba et al. under review). Pseudosuccinea columella shows no preferences for anthropic or natural habitats [START_REF] Vázquez | Distribución y preferencia de hábitats de moluscos hospederos intermediarios de Fasciola hepatica en Cuba[END_REF]. Considered to have a secondary role as intermediate host of F. hepatica in Cuba [START_REF] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF], only two populations, located in Pinar del Rio province (i.e. IPA and Pilon, western Cuba), have been found naturally-infected with F. hepatica, with prevalence of 3 and 10%, respectively (Alba et al. under review, Gutiérrez et al., 2011). Despite so relatively few natural infections with F. hepatica as compared to G. cubensis, snails from most of the P. columella field populations can be successfully infected in the laboratory with Cuban F. hepatica isolates [START_REF] Calienes | Detection and genetic distance of resistant populations of Pseudosuccinea columella (Mollusca: Lymnaeidae) to Fasciola hepatica (Trematoda: Digenea) using RAPD markers[END_REF][START_REF] Gutiérrez | Fasciola hepatica (Trematoda: Digenea): its effect on the life history traits of Pseudosuccinea columella (Gastropoda: Lymnaeidae), an uncommon interaction[END_REF][START_REF] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF]. The prevalence of infection following lab-exposure of P. columella to F. hepatica varies depending on the snail (population) -parasite (isolate) combination used, indicating the existence of a polymorphism of compatibility in this parasite -host system [START_REF] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF][START_REF] Alba | Fasciola hepatica -Pseudosuccinea columella interaction: effect of increasing parasite doses, successive exposures and geographic origin on the infection outcome of naturally-resistant and susceptible snails from Cuba[END_REF].

Remarkably, P. columella snails from particular localities on Cuba, i.e. La Palma, El Azufre, Babiney, La Playita, La Coca and Candelaria (see Alba et al. (under review)) have never been found with naturally occurring F. hepatica infection, nor do they incur infection after experimental exposures to field-derived parasites, regardless of the local F. hepatica isolates used, challenge with increasing doses of the infective larvae (from 5 to 30 miracidia/snail), through single or serial exposures, and with allopatric parasites from Dominican Republic and from France [START_REF] Alba | Fasciola hepatica -Pseudosuccinea columella interaction: effect of increasing parasite doses, successive exposures and geographic origin on the infection outcome of naturally-resistant and susceptible snails from Cuba[END_REF][START_REF] Calienes | Detection and genetic distance of resistant populations of Pseudosuccinea columella (Mollusca: Lymnaeidae) to Fasciola hepatica (Trematoda: Digenea) using RAPD markers[END_REF][START_REF] Gutiérrez | Fasciola hepatica (Trematoda: Digenea): its effect on the life history traits of Pseudosuccinea columella (Gastropoda: Lymnaeidae), an uncommon interaction[END_REF]Gutiérrez et al., , 2003b;;[START_REF] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF]. A previous study informed that the immune cells (hemocytes) of these P. columella snails encapsulate F. hepatica larva shortly after parasite penetration (at 24h post-exposure; Gutiérrez et al., 2003b). The above results indicate the occurrence of an effective immunological response rather than an unsuitability of these snails to F. hepatica [START_REF] Alba | Fasciola hepatica -Pseudosuccinea columella interaction: effect of increasing parasite doses, successive exposures and geographic origin on the infection outcome of naturally-resistant and susceptible snails from Cuba[END_REF]Gutiérrez et al., 2003b;[START_REF] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF]. Interestingly, as with F. hepatica-susceptible P. columella successful natural and experimental infections of resistant snails with other trematodes occur suggesting a certain specificity of the resistance (authors' unpublished data).

One species, two phenotypes

Significantly, resistant and F. hepatica-susceptible P. columella snails show no differences concerning the anatomy of their reproductive system, and share the same reliable conchological characters that are defined for P. columella: big shell, small spire, large aperture of about two thirds of the shell height and characteristic micro-sculptures in the periostracum [START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF][START_REF] Pointier | Guide to the Freshwater Molluscs of the Leasser Antilles[END_REF]Vázquez and Sánchez, 2015). In addition, amplification and sequencing of nuclear ribosomal genes (1170 bp comprising the 3′ region of the 18S, ITS-1, 5.8S, ITS-2 and the 5' region of the 28S) from resistant and susceptible snails showed a slight difference between phenotypes in only two bases, one in each ITS fragment (Gutiérrez et al., 2003a). Such differences represented only 0.17% of sequence variation between the two strains of P. columella and thus, are insufficient to segregate both phenotypes into different species (Gutiérrez et al., 2003a) especially considering the high level of selffertilization observed in this species [START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at worldwide scale[END_REF]. All the above criteria can be used to discriminate certain species of molluscs, even within the family Lymnaeidae (e.g. P. columella; [START_REF] Bargues | Phylogenetic analysis of Lymnaeid snails based on 18S rDNA sequences[END_REF][START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF] but only when differences are evident. The high similarity encountered between these strains at morphologic, anatomic and genetic levels points at the occurrence of two different phenotypes concerning F. hepatica infection within P. columella species (Gutiérrez et al., 2003a). To our knowledge, this is a unique report of field-occurring resistance/susceptibility in a snail -trematode system with perhaps, the exception of the antidigenean persistent resistance in the snail isolate BS90 of Biomphalaria glabrata versus Schistosoma mansoni [START_REF] Paraense | Variation in susceptibility of populations of Australorbis glabratus to a strain of Schistosoma mansoni[END_REF].

It is important to mention that the inheritance pattern of the specific markers concerning susceptibility and resistance of P. columella remains to be established. However, this issue is difficult to resolve by mating experiments as it has been demonstrated, by microsatellite markersbased analysis, that self-fertilization constitutes the almost exclusive reproductive strategy of this species, irrespective of its phenotype (Alba et al. under review, Lounnas et al., 2017;[START_REF] Nicot | Characterization of 15 microsatellite loci in the pulmonate snail Pseudosuccinea columella (Mollusca, Gastropoda)[END_REF]. A highselfing rate is, in fact, a common feature of the family Lymnaeidae and has been reported also for other species including G. truncatula, the main vector of fasciolosis worldwide [START_REF] Meunier | Field and experimental evidence of preferential selfing in the freshwater mollusc Lymnaea truncatula (Gastropoda, Pulmonata)[END_REF].

Morphological and genetic differences between P. columella Phenotypes

The resistant and susceptible P. columella populations can be readily and reliably discriminated by a morphological marker consisting of characteristic pigmentation pattern of the mantle (Gutiérrez et al., 2003b). All resistant snails display a band of small sharp spots in the mid-region of the mantle with bigger spots uniformly distributed on the upper and lower sides (Alba et al. under review, Gutiérrez et al., 2003b). Contrastingly, susceptible snails have diffused and sparser mantle spots, scattered without a clearly defined pattern (Gutiérrez et al., 2003b; see Fig. 1 for details).

Resistant populations of P. columella can be also distinguished from Susceptible snails by randomly amplified polymorphic DNA; RAPD [START_REF] Calienes | Detection and genetic distance of resistant populations of Pseudosuccinea columella (Mollusca: Lymnaeidae) to Fasciola hepatica (Trematoda: Digenea) using RAPD markers[END_REF]Gutiérrez et al., 2003a), mitochondrial haplotypes [START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at worldwide scale[END_REF] and nuclear microsatellites (Alba et al. under review;[START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at worldwide scale[END_REF]. These studies have demonstrated that resistant populations clustered separately from most P. columella populations from Cuba (Alba et al. under review, Calienes et al., 2004) and from the rest of the world [START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at worldwide scale[END_REF], supporting the notion of a genetic determinism of the resistance to F. hepatica. Such segregation suggests that resistant P. columella have been selected from different pools than the susceptible snails that have spread widely, and that P. columella snails grouped outside of the "resistant" cluster could be primarily thought as susceptible to F. hepatica. Additionally, resistant populations (i.e: La Palma, El Azufre, Babiney and La Coca), showed higher overall allelic richness and a marked differentiation of microsatellites-based genetic population structure (Alba et al. under review;[START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at worldwide scale[END_REF] compared to susceptible P. columella from Cuba that were determined to be predominantly monomorphic (Alba et al. under review). These findings point to a more ancient introduction of the resistant populations and a detached evolutionary history from the susceptible phenotype in Cuba (Alba et al. under review). However, despite the indications of an earlier arrival on Cuba, resistant P. columella present a very discrete geographical distribution (only six localities listed above) compared to susceptible populations, and this suggest the existence of an ecological cost of the resistance that constraints range expansion in nature (Alba et al. under review).

Ecological patterns associated to the resistant phenotype: fitness cost of resistance

A follow-up study on snail abundance conducted in two nearby water bodies harboring either resistant (El Azufre) or susceptible (IPA) P. columella populations showed stable densities for both populations throughout the year. However, abundance of resistant P. columella was always lower compared to the susceptible population (Gutiérrez et al., 2005a).

Recently, ecological patterns associated with the distribution of each phenotype in nature have been analyzed separately for resistant and susceptible P. columella populations (Alba et al. under review). It was determined that while susceptible and resistant snails share similar ecological requirements, resistant populations only occurred in sites with low richness of other snail species (3.2 ± 1.02), characterized by slightly acid (pH=6-6.5) and soft (total hardness (TH)=4°-10°d) water (Alba et al. under review). Experiments in the laboratory showed that lowered pH/TH water conditions negatively affect P. columella species regardless of the phenotype, but resistant strains were significantly more tolerant to such conditions, as evidenced by higher survival rates, and both greater life expectancy and percentage of viable eggs as compared to susceptible strains (Alba et al. under review).

However, lower fecundity rates, delay in egg hatching, late reproductive peaks (Alba et al. under review) and diminished net reproductive rate [START_REF] Gutiérrez | Fasciola hepatica (Trematoda: Digenea): its effect on the life history traits of Pseudosuccinea columella (Gastropoda: Lymnaeidae), an uncommon interaction[END_REF] are indistinctively reported in resistant P. columella isolates suggesting the occurrence of a certain trade-off against reproduction. Particularly, resistant snails (i.e. La Palma) incur a diminished net reproductive rate if they are reared in the presence of susceptible P. columella. By contrast, susceptible snails raised in competition with resistant individuals increase their shell growth and net reproductive rate (Gutiérrez et al., 2005b). These results have led to the following hypothesis to explain the restricted distribution of resistant P. columella in nature. It is postulated that the existence of an ecological cost of the resistance, possibly manifested as reproductive impairments, results in a less competitive potential of resistant compared to susceptible snails (Alba et al. under review).

However, the higher tolerance to environmental pH/TH stress likely provide the resistant phenotype with an ecological advantage in sites with lower pH/TH conditions that are less suitable for other snails, and provide a concomitantly reduced competition from other (species of) snails (Alba et al. under review).

Overall, regarding life traits following experimental challenge with F. hepatica, exposed resistant snails exhibited higher survival rates than infected-susceptible isolates [START_REF] Alba | Fasciola hepatica -Pseudosuccinea columella interaction: effect of increasing parasite doses, successive exposures and geographic origin on the infection outcome of naturally-resistant and susceptible snails from Cuba[END_REF][START_REF] Gutiérrez | Fasciola hepatica (Trematoda: Digenea): its effect on the life history traits of Pseudosuccinea columella (Gastropoda: Lymnaeidae), an uncommon interaction[END_REF].

In addition, no significant variations were observed in the fecundity rates between non-exposed and exposed resistant snails [START_REF] Gutiérrez | Fasciola hepatica (Trematoda: Digenea): its effect on the life history traits of Pseudosuccinea columella (Gastropoda: Lymnaeidae), an uncommon interaction[END_REF].

Comparative molecular screening identifies differences between resistant and susceptible P. columella

An understanding of the fine mechanisms that mediate P. columella resistance to F. hepatica is of interest for the development of new strategies aiming at unveiling and controlling F. hepatica transmission but also for study of determinants of pathogen virulence and for comparative immunology. However, despite the gathering of extensive phenotypical, genetic and ecological data, no study has been conducted to decipher the molecular scenario that give rise to each distinct P. columella phenotype relative to F. hepatica. To undertake this task, we sought to investigate differences between naïve susceptible and resistant P. columella at both the transcriptome and proteome levels.

Firstly, we constructed a de novo assembled transcriptome for this species from whole snails by RNAseq and performed comparative analysis to identify differential patterns of abundance of transcripts that may possibly account for the phenotypic and ecological features of P. columella resistance to F. hepatica and tolerance to pH/TH stress.

Secondly, we performed 2D-electrophoresis, and MS/MS spectrometry, to identify differentially expressed proteins in a particular anatomical compartment of the female snail reproductive system, the albumen gland. Apart from its role of producing the perivitelline fluid that is deposed in eggs to nurture the embryos [START_REF] Duncan | Pulmonates[END_REF][START_REF] Geraerts | Freshwater snails (Basommatophora)[END_REF], the albumen gland is also involved in the synthesis of a number of immune effectors and defense proteins [START_REF] Guillou | Identification and expression of gene transcripts generated during an anti-parasitic response in Biomphalaria glabrata[END_REF][START_REF] Hathaway | Identification of protein components of egg masses indicates parental investment in immunoprotection of offspring by Biomphalaria glabrata (Gastropoda, Mollusca)[END_REF][START_REF] Mu | An integrated proteomic and transcriptomic analysis of perivitelline fluid proteins in a freshwater gastropod laying aerial eggs[END_REF][START_REF] Sen | Albumen gland of the snail Achatina fulica is the site for synthesis of Achatinin H, a sialic acid binding lectin[END_REF]. Therefore, comparative analysis of the proteome of this multifunctional organ can give insights into the suggested trade-off between resistance to F. hepatica and reproduction in resistant snail isolates (Gutiérrez et al., 2002, Alba et al. under review).

In this study, we found similar patterns from the transcriptomic and the proteomic approaches: a significant bias towards an overall increased abundance of defense and stress-related process transcripts/ proteins was associated with resistant snails versus the F. hepatica-susceptible phenotype of P. columella. These results support self-maintenance as favored strategy of resistant P. columella. Differences in metabolic organization to the detriment of protein synthesis/folding was also observed from both "omic" analyses. This may be particularly significant in the case of the albumen gland, potentially contributing to the reproductive constraints observed in resistant P. columella populations. The detected differences in expression are discussed in regard to the differential phenotypic and ecological features of the resistant and susceptible strains. In addition, this paper presents a list of candidate factors for further functional validation as predictors of susceptibility to infection and/or for potential inroads toward control of infection and transmission of fascioliasis by boost snail immune function.

Material and methods

Laboratory-reared P. columella snails

Five-week-old P. columella snails (average size 5.09 ± 0.64 cm) from F. hepatica-resistant (La Coca) and -susceptible (Aurora) populations, reared in the Laboratory of Malacology of the IPK (see [START_REF] Sánchez | Cultivo de Fossaria cubensis (Pfeiffer) (Pulmonata: Lymnaeidae) hospedero intermediario de Fasciola hepatica (Linnaeus) en Cuba[END_REF] for details on rearing) were used for the transcriptomic and the proteomic experiments. To gain an overview on constitutive differences between phenotypes, only naive snails (non-exposed to parasite infection) were sampled.

Comparative transcriptomic approach on whole snail 2.2.1. RNA extraction and sequencing by Illumina

Sixty P. columella snails per strain were used to obtain three biological replicates of 20 snails each. Briefly, snails were separated from the shell and immediately placed in individual vials containing RNAlater® (Ambion) at a ratio 10 μL solution to 1 mg of snail tissue. Samples were kept overnight at 4 °C and then stored at -20 °C until use. For RNA extraction, RNAlater® was discarded and whole-tissues of 20 snails per strain (three biological replicates per strain) were pooled, immediately frozen in liquid nitrogen and homogenized. RNA extraction was performed with TRIzol® Reagent (Ambion) following manufacturer's guidelines. Extracted RNA was purified using RNeasy® Minikit (Qiagen) and treated with DNase following the in-column procedure of Turbo DNA-freeTM kit (Ambion). The quality and quantity of the extracted RNA was assessed on a 2100 Bioanalyzer using an RNA 6000 Nano kit (Agilent Technologies) and by Qubit™ 2.0 fluorometry (Invitrogen).

One μg of total RNA from each biological replicate was used for library preparation using Illumina TruSeq stranded mRNA kit and RNA sequencing was performed by Fasteris SA (Geneva, Switzerland), collecting paired-end, 75-bp read length reads, with three samples multiplexed per lane, using the Illumina HiSeq™4000 platform.

De novo assembly and annotation of P. columella transcriptome

A de novo transcriptome of P. columella was assembled using highquality reads (quality > 38 phred score) from all six sequenced samples using the default options (which includes the normalization step) of Trinity 2.0.6.1 method [START_REF] Grabher | Full-length transcriptome assembly from RNA-Seq data without a reference genome[END_REF] on Galaxy Project Server [START_REF] Giardine | Galaxy: a platform for interactive large-scale genome analysis[END_REF] at the instances of the IHPE laboratory (http://bioinfo.univ-perp.fr). From a total of 592 million raw reads, the first consensus transcriptome for P. columella resulted into 158 837 contigs, named full-transcriptome. To reduce its complexity, Trinity Super Assembly [START_REF] Grabher | Full-length transcriptome assembly from RNA-Seq data without a reference genome[END_REF] was applied to the de novo assembled transcriptome, and all transcripts shorter than 300 bp were removed, with 78 774 transcripts remaining for further analysis.

Afterwards, as two phenotypes (and populations) with genetic differences within P. columella were included for RNAseq (see Alba et al. under review;[START_REF] Calienes | Detection and genetic distance of resistant populations of Pseudosuccinea columella (Mollusca: Lymnaeidae) to Fasciola hepatica (Trematoda: Digenea) using RAPD markers[END_REF][START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at worldwide scale[END_REF], hypervariable families were reduced by CD-Hit-est [START_REF] Li | Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences[END_REF]) by clustering transcripts with matches above 95% identity. This results in 72748 transcripts, designated by the name simplified transcriptome.

We used BUSCO tool (available http://busco.ezlab.org/v1; [START_REF] Simão | BUSCO: ssessing genome assembly and annotation completeness with single-copy orthologs[END_REF] to assess the quality and completeness of the assembled transcriptomes. We obtained 909 out of 978 complete BUSCOs; no information losses resulted from the reduction steps. All RNAseq data is available in the Sequence Read Archive of NCBI under the following accession numbers: Submission number SUB5947916; BioProject: PRJNA555222: Pseudosuccinea columella RNA sequencing; BioSample: SAMN12305757: Pseudosuccinea columella RNAseq (TaxID: 31228). Transcriptomes are also available at the laboratory database: http://ihpe.univ-perp.fr/acces-aux-donnees/.

The automatic annotation of the simplified transcriptome was performed by Blast2GO version 2.4.2 [START_REF] Conesa | Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research[END_REF] using BLASTx against the National Center for Biotechnology Information (NCBI) nonredundant (nr) sequences database (with an E-value threshold set at 1E-03) was performed. Gene function, protein domain and enzyme annotation were also assigned by similarity searches using the Gene Ontology (GO) database and InterPro scan. Annotation results were obtained for only 38 968 transcripts (49.5%).

Comparative analysis between resistant and susceptible P. columella

For comparative analysis between susceptible and resistant P. columella phenotypes, quality reads (phred score > 38) were aligned to the reduced assembled transcriptome using Bowtie2 v 2.0.2 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] set in fast end-to-end mode. Transcripts were counted with Salmon [START_REF] Patro | Salmon provides fast and biasaware quantification of transcript expression[END_REF] and differential expression was calculated by DESeq2 [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF], accounting for over or under-representation in the resistant phenotype. As threshold criteria, only differentiallyexpressed transcripts with adjusted P < 0.05 and displaying more than 2-fold changes were considered. All RNAseq analyses were run locally using the Galaxy Project server [START_REF] Giardine | Galaxy: a platform for interactive large-scale genome analysis[END_REF].

Enrichment analyses of the transcriptome involving biological processes or molecular functions, considering separately over-and under-expressed transcript data, were performed using Fisher's Exact Test run on Blast2GO software. A particular GO term with P and false discovery rate (FDR) < 0.05 was considered enriched. In addition, differentially-expressed transcripts were evaluated combining Gene Ontology (GO) terms and keyword list of well-known defense molecules to select the genes putatively involved in immunity (i.e. defense response, wound healing and inflammation) and in relation to acidbase balance (i.e. carbon-bicarbonate buffering and ion transportation).

Comparative proteomic approach on the albumen gland 2.3.1. Protein extraction of albumen glands

The albumen gland of 15 naive snails from each strain (biological replicates) was removed by dissection, lyophilized and individually processed. Briefly, the crude extract of the albumen gland from each individual snail was obtained by sonication on ice at 20% amplitude (Vibra Cell 75185 sonicator; Bioblock Scientific) for 20 s in 80 μL of icecold denaturing buffer (urea 7 M, thiourea 2 M, CHAPS 4% in Tris-HCl 30 mM, pH 8.5) and clarified by centrifugation at 2000 x g, 15 min, 4 °C. Protein concentration of the supernatant was estimated using the 2D Quant Kit (GE Healthcare) and samples were stored at -80 °C until used.

2D gel electrophoresis of protein extracts from P. columella albumen Glands

Proteins extracted from each albumen gland were individually analyzed by 2D gel electrophoresis (15 gels per strain, each corresponding to different biological replicates). One-hundred μg of proteins from each extract were added to rehydration buffer (urea 7 M, thiourea 2 M, CHAPS 4%, DTT 65 mM) for a total volume of 350 μL. Individual samples were loaded onto 17 cm isoelectric focusing strips (BioRad) with a stabilized non-linear pH ranging from 3 to 10. Passive strip rehydration was achieved by 5h at 22 °C, followed by an active rehydration of 14 h under a 50 V current (to help big proteins to enter into the strips) at the same temperature. Afterwards, isoelectric focusing of proteins was performed using the following program: 50 V for 1 h, 250 V for 1 h, 8000 V for 1 h and a final step at 8000 V for a total of 90 000 Vh with a slow ramping voltage (quadratic increasing voltage) at each step. Focused proteins were reduced by incubating the strip twice with equilibration buffer (Tris, 1.5 M; urea, 6 M; SDS, 2%; glycerol, 30%; bromophenol blue; pH 8.8) containing DTT (130 mM) at 55 °C. Then, proteins were alkylated by an incubation with equilibration buffer containing iodoacetamide (135 mM) on a rocking agitator (400 rpm) at room temperature protected from light. Proteins were separated according to their molecular weight (second dimension) on 12% acrylamide/0.32% piperazine diacrylamide gels run at 25 mA/gel for 30 min followed by 75 mA/gel for 8 h using a Protean II XL system (Bio-Rad). Gels were stained with MS-compatible silver staining protocol and scanned using a ChemiDoc MP Imaging System (Bio-Rad) associated with Image Lab software version 4.0.1 (Bio-Rad).

Comparative analysis of 2D-gels between susceptible and resistant P. columella

Considering the complexity of the 2D gel profile of albumen gland, and quality of sample resolution, five most representative gels per strain were selected for comparative analysis on PD-Quest v. 7.4.0 (Bio-Rad) to identify differences in protein abundance between proteomic profiles of albumen glands from susceptible versus resistant individual snails. When a spot was reproducibly present in all five replicates from one strain and absent from all five replicates from the other strain, it was considered a qualitative change. For quantitative changes, only spots whose mean intensity across five replicates per strain was four-fold higher or lower than those from the other strain, with a P < 0.01 (Mann-Whitney U test), were considered as significantly differentially abundant between the two strains. Selection of spots for further analysis by MS/MS spectrometry was based on qualitative (i.e. present in only one of two phenotypes) or highly quantitative differences (i.e.>4-fold differentially abundant; P < 0.01). Differentially represented spots were excised from the gels, destained, trypsin digested and the obtained peptides were identified by tandem mass spectrometry using the PISSARO platform facility (University of Rouen, France).

For the identification of the protein(s) present in each spot, the obtained peptides were matched to the consensus de novo assembled full transcriptome for P. columella (158837 transcripts). The transcript sequences confidently matching the peptides were used as a query for a xBLAST against nonredundant NCBI database to determine the protein identity of the best match. Up to the first three best hits were considered, when at least 4 peptides matched the sequence with a coverage> 10%. Identified transcripts sequences were translated into protein using Expasy server [START_REF] Gasteiger | ExPASy: the proteomics server for in-depth protein knowledge and analysis[END_REF] to identify conserved domains with CDD [START_REF] Marchler-Bauer | CDD: NCBI's conserved domain database[END_REF]. The theoretical pI and molecular weight were also calculated using the Expasy server [START_REF] Gasteiger | ExPASy: the proteomics server for in-depth protein knowledge and analysis[END_REF] to cross-reference the protein sequence data with the location of the spot on the gel. Altogether, these complementary analyses allowed confident characterization of the protein identity of each spot.

Results

Transcriptomic differences between susceptible and resistant P. columella phenotypes

Overall, 97.49% of the Illumina RNAseq reads from both phenotypes of P columella were successfully realigned against the simplified transcriptome. A total of 6876 transcripts (9.45%; 4126 up-regulated and 2750 down-regulated) showed a significantly greater than 2-fold differentialexpression between phenotypes (see supplementary data 1) from which 3290 transcripts (47.5%) had no annotation results.

Results from the enrichment analyses for both over-and under-represented transcripts (test data) relative to the annotated simplified transcriptome (reference data) are shown in Fig. 2 and supplementary data 2. Analysis of the increased expression transcript data set showed that the resistant phenotype associated with five main categories of biological processes: biological regulation and homeostasis, defense/ stress responses, primary signaling pathways/transduction, and nitrogen metabolism, particularly biosynthesis, maintenance and repair of DNA (Fig. 2A). In addition, molecular functions related to signal transduction (e.g. transmembrane receptor/G-protein coupled receptor and protein tyrosine phosphatase activities) and with Ca2+ binding were enriched in the resistant isolate (Fig. 2B). Contrastingly, metabolic process that were related to protein synthesis were underrepresented in resistant snails (e.g. ribosome biogenesis, structural constituent of ribosome, peptide biosynthetic process, translation; Fig. 2C).

It was particularly significant to find an enrichment in biological processes related to defense and stress response in the resistant phenotype (see Fig. 2A) supported by increased abundance in resistant snails of transcripts for pathogen receptor/interacting molecules, transcripts with regulatory roles in immunity and molecules involved in the activation and orchestration of defense responses (see Fig. 3 A,B; supplementary data 1). These included several lectins, CD109, cytokines and cytokinerelated molecules (e.g. macrophage migration inhibitory factor (MIF), granulocyte colony stimulatory factor receptor (G-CSFR), transforming growth factor 1-beta (TGF1β)), as well as of signaling/regulatory transcripts (e.g. Toll-like receptors (TLR), protein C kinase (PKC), members of the superfamily of tumoral necrosis factor (TNF) receptors and interferon regulatory factors (IRF), see (Fig. 3B).

Additionally, potential anti-parasitic defenses like ferritin, nitric oxide synthase and antioxidant molecules such as catalase, superoxide dismutase (SOD) and probable deferrochelatase peroxidase were also more abundant in resistant snails (Fig. 3B). Increased levels of immune surveillance in the resistant phenotype were suggested by transcripts that can function in leukocyte adhesion, rolling and tethering (interference hedgehog-like, Ras-like GTP-binding Rho1, rho-associated kinase 1), and wound healing (Ras-like GTP-binding Rho1).

Concordant with the association of greater tolerance for variations in pH and TH levels with snails from resistant P. columella populations (Alba et al. under review), several major ion regulatory factors, and other transporters that contribute to pH and osmotic regulation were observed more from resistant snails (Fig. 3C; supplementary data 1). In addition, carbonic anhydrase transcripts were also found highly abundant in this isolate (Fig. 3C).

Qualitative and quantitative differences between proteomes of albumen glands from susceptible and resistant P. columella phenotypes

Albumen glands of P. columella exhibited complex proteomic profiles notably consisting of highly abundant proteins of high molecular weight and acidic pI (upper left part of the gels in Fig. 4). Such highly abundant proteins with several isoforms can mask nearby proteins and impede the quantitative analysis and proper protein identification. Therefore, this region of the gels, identified with a white rectangle in Fig. 4, was excluded from analysis. A total of 554 spots were identified from both proteomes, from which more than 80% were present and similarly abundant in both phenotypes.

However, 18 spots were uniquely to resistant snails while 13 were only observed from susceptible individuals (supplementary data 3; Fig. 4). Twenty-eight spots differed significantly (P < 0.01, Mann-Whitney U test,>4-fold) in abundance between resistant and susceptible P. columella (Supplementary data 3; Figs. 4), 12 and 16 spots of the resistant strain were over-and underrepresented, respectively compared to the susceptible strain (Supplementary data 3; Fig. 4). The ratios of resistant/susceptible quantity ranged from 0.24 (spot # 2107) up to 26.11 (spot # 7117).

The selection of 59 spots representing differentially expressed proteins for MS characterization yielded successful identification of 49 proteins and several isoforms (Table 1; Supplementary data 3). No identification was made from spots 7107, 7108 and 6515. Spot 6002 contained two protein isoforms with 67% sequence similarity (Evalue=2E-67 in BLASTp output; Supplementary data 3) to an uncharacterized protein (LOC106073623) predicted from genome sequence of B. glabrata snails (NCBI Biomphalaria glabrata Annotation Release 100), which has been associated with certain lysozyme and peptidoglycan binding activities according to BLASTp consensus [START_REF] Buddenborg | Transcriptomic responses of Biomphalaria pfeifferi to Schistosoma mansoni: investigation of a neglected African snail that supports more S. mansoni transmission than any other snail species[END_REF]. However, no putative conserved domains were detected in the isoforms of spot 6002 (Supplementary data 3). The presence of more than one protein or of different protein isoforms into the same spot in the same gel was expected due to the complexity of the electrophoretic profiles (see Fig. 4).

Table 1 summarizes the proteins (from one up to four hits per spot) That were identified by MS analysis from the 59 spots excised from the 2D gels. Overall, defense/stress and metabolism-related molecules were more abundant in resistant snails (Table 1). Particularly, G-type lysozyme and lipopolysaccharide-binding protein/bactericidal permeability-increasing protein (LBP/BPI) were only observed in resistant snails (Table 1). Conversely, proteins related to protein anabolism were underrepresented in resistant compared to susceptible snails (Table 1).

It is worth noting some qualitative differences between isolates that concern the proteins that were identified as involved in signaling processes. In this sense, abundance of tyramine β-hydrolase like (TBH) in resistant snails, an enzyme that catalyzes the conversion of the neurotransmitter tyramine into octopamine, contrasts with the lower representation of dopamine β-hydrolase-like monooxygenase (which mainly converts dopamine in norepinephrine) as compared with susceptible snails (Table 1).

Discussion

Omics provide a molecular baseline for phenotypic and ecological features of P. columella snails that are naturally resistant to F. hepatica 4.1.1. Molecular clues for parasite resistance and high tolerance to pH/TH variations

Resistance to F. hepatica in P. columella has been associated with an active encapsulation of the invading parasite shortly after penetration that develops fully within 24h post-exposure and that eventually leads to the death of the larvae and to the resolution of the infection (Gutiérrez et al., 2003b). The consistent nature of the rapid commitment to this immune response in resistant P. columella, and the lack thereof in snails from susceptible populations reasonably support the thought that some features involved in the susceptibility/resistance to the parasite F. hepatica are constitutively evident in each snail phenotype. In the present paper, both mRNA-and protein-centered "omic" approaches showed an overall enrichment in resistant snails, of biological functions associated with defense and stress responses. It is worth to mention that proteomic differences could be more extensive, due to the exclusion of the top left region of gels (lots of large proteins) from the study. However, these finding are possibly related to the fact that both defense and stress responses are linked by several molecular functions, effectors and signaling pathways [START_REF] Demas | Neuroendocrine-immune crosstalk in vertebrates and invertebrates: implications for host defence[END_REF][START_REF] Matozzo | First evidence of immunomodulation in bivalves under seawater acidification and increased temperature[END_REF]. In fact, both the effective hemocytic encapsulation of F. hepatica (Gutiérrez et al., 2003b) and the higher tolerance to pH/TH variations associated with resistant P. columella (Alba et al. under review) are responses toward (biotic or abiotic) stressors.

On the one hand, the greater abundance of immune-related transcripts/proteins (e.g. several lectins, pro-inflammatory signaling pathways, cytokines, effector molecules) compared to susceptible snails, may provide significant advantage to resistant snails, enabling the accelerated mounting and regulating of an early and efficient immune response to F. hepatica. Similarly, elevated constitutive molecular processes were proposed to be the base for snail refractoriness to parasite immunosuppressive factors in B. glabrata strains, experimentally-selected for resistance to Echinostoma caproni [START_REF] Humbert | Refractoriness of host haemocytes to parasite immunosuppressive factors as a putative resistance mechanism in the Biomphalaria glabrata-Echinostoma caproni system[END_REF]. Notably, representation of plasma proteins differed significantly in these susceptible and resistant B. glabrata strains and was linked to a differential gene regulation expressed in the albumen gland [START_REF] Vergote | Characterisation of proteins differentially present in the plasma of Biomphalaria glabrata susceptible or resistant to Echinostoma caproni[END_REF].

On the other hand, overall higher abundance in resistant snails of several carbonic anhydrase isoforms, both at the transcriptomic or the proteomic level (see Fig. 3C, Table 1) suggests a higher potential to compensate for pH variations [START_REF] Freitas | Biochemical alterations induced in Hediste diversicolor under seawater acidification conditions[END_REF]. Likewise, given that water acidification negatively affects osmotic regulation in aquatic invertebrates [START_REF] Freitas | Biochemical alterations induced in Hediste diversicolor under seawater acidification conditions[END_REF][START_REF] Mattews | Acid-base regulation in insect hemolymph[END_REF], constitutive over-representation of different ion regulators and transporters (see Fig. 3C) may, in part, provide the molecular bases the for increased tolerance to pH/TH stress that was observed from resistant P. columella (Alba et al. under review).

Furthermore, there was enrichment in molecular functions related to Ca 2 + binding. This ion is a common secondary messenger inside the cells with important roles in signal transduction during several processes including immune response in molluscs [START_REF] Tunholi-Alves | Changes in the calcium metabolism of Biomphalaria glabrata experimentally infected with Angiostrongylus cantonensis[END_REF]. In addition, it is essential for shell formation and alteration of calcium levels in hemolymph during infection or due to pH regulation can result in shell hypocalcification [START_REF] Tunholi | Influence of Echinostoma paraensei (Lie and Basch, 1967) infection on the calcium content in Biomphalaria glabrata (Say, 1818)[END_REF][START_REF] Tunholi | Biological, biochemical and histological features of Bradybaena similaris (Gastropoda: Pulmonata) infected by Heterorabditis indica (Rhabditida: Heterorhabditidae) strain LPP1[END_REF][START_REF] Tunholi-Alves | Changes in the calcium metabolism of Biomphalaria glabrata experimentally infected with Angiostrongylus cantonensis[END_REF]. Thus, it is hypothesized here that enhanced capabilities to manage calcium balance is also linked with the resistance to F. hepatica and the tolerance to pH/TH stress in P. columella, and might be somehow related to the characteristic pigmentation patterns displayed in the mantle of resistant snails as it is the site of shell formation (see Gutiérrez et al. (2003b)).

Resistance trade-off against reproduction: molecular clues from the albumen gland

The differential transcription effort towards stress/defense response observed in naïve resistant P. columella suggests that self-maintenance and survival is the preferred life strategy in this phenotype. However, energy budget constraints cause trade-off against other biological processes, as exemplified by reproductive constraints observed in some pathogen-resistant hosts, (e.g. [START_REF] Langand | Cost of resistance, expressed as a delayed maturity, detected in the host-parasite system Biomphalaria glabrata/Echinostoma caproni[END_REF]. The limited reproductive output specifically of the resistant phenotype of P. columella (Alba et al. under review) may be associated with the underrepresentation of biological process/functions/proteins related to protein synthesis, evident from both "omic" approaches (see Fig. 2C and Table 1). Enhanced immune responsiveness may reconfigure intermediate metabolism towards a relative increase of the respiration rate, glycolysis, proteolysis and lipolysis [START_REF] Lochmiller | Trade-offs in evolutionary immunology: just what is the cost of immunity?[END_REF], and increased defenses against stress (e.g. antioxidative enzymes [START_REF] Freitas | Biochemical alterations induced in Hediste diversicolor under seawater acidification conditions[END_REF] may cause lower energy reserves (protein and glycogen content) and higher metabolic rates.

In the albumen gland of resistant snails, energy production through the respiratory chain and energy storage (synthesis of fatty acid) were indicated particularly active by the abundance of cytochrome b-c1 complex subunit 2 mitochondrial-like isoforms and of 3-oxoacyl-reductase FabG-like (catalyzes the first step of the fatty acid synthesis), respectively (see Table 1). Elevated representation of 4-hydroxyphenylpyruvate dioxygenase-like may indicate an association with catabolism of aromatic amino acids, whose intermediates enter into the Kreb's cycle. These processes likely constrain the production of the protein components of the perivitelline fluid, that are normally deposited in the eggs and directly involved in the sustainability of the embryos, a primary reproductive function of the albumen gland [START_REF] Duncan | Pulmonates[END_REF][START_REF] Geraerts | Freshwater snails (Basommatophora)[END_REF]. Consequently, the reproductive output of resistant snails, whether in quantity (fecundity rates, net reproductive rate), quality (viable eggs) or in time (late reproductive peaks), might be affected especially because the albumen gland is also devoted to the synthesis of proteins involved in immunity and other functions (see Table 1). This may then results in a trade-off against reproduction, particularly egg production/viability. Thus, results from this study may provide, at least in part, insights into the molecular basis of the reproductive constraints of resistant compared to susceptible snails (see Alba et al. (under review); [START_REF] Gutiérrez | Fasciola hepatica (Trematoda: Digenea): its effect on the life history traits of Pseudosuccinea columella (Gastropoda: Lymnaeidae), an uncommon interaction[END_REF] for details).

Speculatively, the metabolic rearrangement and allocation of resources in the albumen gland of resistant snails, as proposed here, may relate to differences in neurotransmitter metabolism observed between P. columella phenotypes (see signaling proteins in Table 1). The presence of TBH suggests that tyramine and, mainly, octopamine arepreferentially involved in neuroendocrine control of the albumen gland of resistant snails. To date, however, only a few studies (from bivalves) inform regarding a functional role of octopamine in molluscan reproduction [START_REF] Blais | Characterization of a novel octopamine receptor expressed in the surf clam Spisula solidissima[END_REF]. Yet other studies refer to potential involvement of octopamine in regulation of feeding and locomotion in gastropods [START_REF] Ormshaw | Octopamine boosts snail locomotion: behavioral and cellular analysis[END_REF][START_REF] Vehovszky | Activation and reconfiguration of fictive feeding by the octopamine-containing modulatory OC interneurons in the snail Lymnaea[END_REF][START_REF] Wentzell | Comparative neurobiology of feeding in the opisthobranch sea slug, Aplysia, and the pulmonate snail, Helisoma: evolutionary considerations[END_REF] and, octopamine participates in the regulation of metabolism, ovulation and egg laying in insects and nematodes [START_REF] Monastirioti | Distinct octopamine cell population residing in the CNS abdominal ganglion controls ovulation in Drosophila melanogaster[END_REF][START_REF] Monastirioti | Characterization of Drosophila tyramine betahydroxylase gene and isolation of mutant flies lacking octopamine[END_REF][START_REF] Tao | Octopamine connects nutrient cues to lipid metabolism upon nutrient deprivation[END_REF]. Additionally, this neurotransmitter was also associated with stress responses in invertebrates. Increased TBH concentrations may be a stress marker in insects [START_REF] Châtel | Characterization of tyramine β-hydroxylase, an enzyme upregulated by stress in Periplaneta americana[END_REF]. In free living nematodes a rise in octopamine levels allows metabolic rearrangements to maintain energy homeostasis during stress induced by starvation [START_REF] Tao | Octopamine connects nutrient cues to lipid metabolism upon nutrient deprivation[END_REF]. Similarly, a significant increase of octopamine has been observed in the central nervous system of severely food-deprived Lymnaea stagnalis snails [START_REF] Aonuma | Weak involvement of octopamine in aversive taste learning in a snail[END_REF]. The results of this study certainly warrant further studies of neurotransmitters concentration and their role in regulating allocation of resources and metabolic functions in the reproductive and immune functions of resistant and susceptible P. columella.

Elevated representation of immune-related molecules in resistant snails

Compared to susceptible snails, naïve resistant P. columella display an overall enhanced immune capacity based on novel and/or elevated expression of an arsenal of defense factors, including pathogen recognition/ interacting molecules, particularly C-type lectins as wells as mannose and galactose binding lectins (see Fig. 3B). The variety of upregulated transcripts potentially able to interact with pathogens displayed by resistant snails may be advantageous given that the first step of any defense response requires recognition of the threat [START_REF] Pinaud | Molecular characterisation of immunological memory following homologous or heterologous challenges in the schistosomiasis vector snail, Biomphalaria glabrata[END_REF]. C-type lectins, in particular, are known for recognition of carbohydrates that constitute pathogen-associated molecular patterns (PAMPs) like LPS and peptidoglycan. Once bound, lectins mediate immune activities ranging from microbe agglutination and opsonization to triggering signaling pathways for induction of phagocytosis and encapsulation [START_REF] Vázquez-Mendoza | Parasitic infections: a role for Ctype lectins receptors[END_REF][START_REF] Pees | High innate immune specificity through diversified C-type Lectin-like domain proteins in invertebrates[END_REF]. In snaildigenean interactions, Ctype lectins from B. glabrata interact with antigens of S. mansoni sporocysts [START_REF] Wu | Proteomic analysis of Biomphalaria glabrata plasma proteins with binding affinity to those expressed by early developing larval Schistosoma mansoni[END_REF] and are expressed by resistant snails in response towards E. caproni infection [START_REF] Guillou | Identification and expression of gene transcripts generated during an anti-parasitic response in Biomphalaria glabrata[END_REF]. Potentially, the great abundance of mannose binding lectins (see Fig. 3B for details) increases the potential of resistant P. columella for rapid effective immune detection of F. hepatica, because the surface of sporocyst is mainly covered with N-acetyl-D-glucosamine and α-mannose [START_REF] Georgieva | Study of surface carbohydrates in Galba truncatula tissues before and after infection with Fasciola hepatica[END_REF]. More effective parasite recognition in resistant P. columella may be contributed to further by highly abundant CD109. The B. glabrata homolog of this thioester-containing protein family member interacts with S. mansoni larvae [START_REF] Wu | Proteomic analysis of Biomphalaria glabrata plasma proteins with binding affinity to those expressed by early developing larval Schistosoma mansoni[END_REF].

Furthermore, abundant transcripts encoding cytokine such as GCSFR, TGF1B and MIF, involved in the proliferation, recruitment and activation of immune cells provide a likely explanation for the effective cellular response that is reliably triggered in resistant snails in response to F. hepatica infection (Gutiérrez et al., 2003b). TGF1B belongs to a family of multifunctional cytokines found in different phyla that share a highly conserved signal transduction pathway and participates in the regulation of different biological functions including stress response and immune modulation [START_REF] Huminiecki | Emergence, development and diversification of the TGF-beta signalling pathway within the animal kingdom[END_REF]. MIF was recorded from several species of mollucs, the homolog of B. glabrata is present in hemocytes, and promotes cell aggregation and hemocyte proliferation, while inhibiting NO-dependent p53-mediated apoptosis. RNAi-mediated knockdown confirmed immune function of B. glabrata MIF, yielding increased parasite burden following exposure to S. mansoni [START_REF] Baeza-Garcia | Involvement of the cytokine MIF in the snail host immune response to the parasite Schistosoma mansoni[END_REF]. Likewise, in Oncomelania hupensis a more distantly related prosobranch gastropod, expression of MIF increased after challenge with Schistosoma japonicum and its involvement in the activation, differentiation and recruitment of hemocytes was demonstrated: RNAi knockdown of MIF decreased the proportion of phagocytic circulating hemocytes and restrains the migration of blood cells from the host towards the site of infection [START_REF] Huang | Identification and functional characterization of Oncomelania hupensis macrophage migration inhibitory factor involved in the snail host innate immune response to the parasite Schistosoma japonicum[END_REF].

The abundance, in resistant snails, of transcripts related to pro-inflammatory signaling pathways (e.g. TLR/Myd88 and PCK) and with regulatory activities (e.g. LRR and IRF) is particularly significant and indicates that increased amounts of signaling components are available for a higher immune responsiveness as is associated with the resistant phenotype. Notably, PKC regulates differentiation and activation of snail hemocytes towards a phenotype more prompt to cellular reactions, particularly promoting increased production of NO and H2O2 levels [START_REF] Humphries | Regulation of hydrogen peroxide release in circulating haemocytes of the planorbid snail Biomphalaria glabrata[END_REF][START_REF] Lacchini | beta-1, 3-glucan modulates PKC signalling in Lymnaea stagnalis defence cells: a role for PKC in H2O2 production and downstream ERK activation[END_REF][START_REF] Wright | Regulation of nitric oxide production in snail (Lymnaea stagnalis) defence cells: a role for PKC and ERK signalling pathways[END_REF] and cell spreading [START_REF] Humphries | Protein kinase C regulation of cell spreading in the molluscan Biomphalaria glabrata embryonic (Bge) cell line[END_REF]. According to [START_REF] Walker | Spreading by snail (Lymnaea stagnalis) defence cells is regulated through integrated PKC, FAK and Src signaling[END_REF], PKC and ERK pathways are involved with focal adhesion kinase in cell adhesion, spreading and formation of lamellipodia, necessary for phagocytosis in L. stagnalis. TLRs are widely studied primary mediators of innate immunity that following recognition of PAMPs, activate highly conserved immune signaling pathways across many different animal phyla. Interestingly, the downstream components (Myd88 and NF-kB) of this signaling pathway were also significantly abundant in resistant snails (see Figs. 2B-3B). In molluscs, TLRs can be activated, and expression up-regulated, after septic injury to promote hemocyte activation (for review see [START_REF] Brennan | Evolutionary origins of Toll-like receptor signaling[END_REF][START_REF] Nie | Toll-like receptors, associated biological roles, and signaling networks in non-mammals[END_REF]. Involvement in snail-trematode interactions was confirmed by a report showing that a TLR was highly expressed in hemocytes from a S. mansoni-resistant strain of B. glabrata and siRNAmediated knockdown significantly reversed the resistant phenotype [START_REF] Pila | A novel Toll-like receptor (TLR) influences compatibility between the gastropod Biomphalaria glabrata, and the digenean trematode Schistosoma mansoni[END_REF].

Other molecules involved in acute phase response and redox killing like ferritin, nitric oxide synthase and antioxidant enzymes are also significantly abundant in resistant snails. These may contribute to the orchestration of effective cellular cytotoxic immune responses that lead to parasite elimination in resistant P. columella. Hemocytes of a B. glabrata strain resistant to S. mansoni highly express ferritin prior parasite infection [START_REF] Lockyer | Early differential gene expression in haemocytes from resistant and susceptible Biomphalaria glabrata strains in response to Schistosoma mansoni[END_REF]. Ferritin binds and regulates iron distribution. As part of an acute phase response, ferritin is upregulated after microbial challenge for iron sequestration in order to deprive infecting microorganisms from iron acquisition and multiplication of pathogens [START_REF] Ong | Iron-withholding strategy in innate immunity[END_REF]. Ferritin also acts as antibacterial agent in mollusks [START_REF] Zheng | Ferritin has an important immune function in the ark shell Scapharca broughtonii[END_REF][START_REF] Chen | Identification and characterization of a ferritin gene involved in the immune defense response of scallop Chlamys farreri[END_REF], remarkably different ferritin isoforms are induced in B. glabrata in a pathogen-specific manner [START_REF] Deleury | Specific versus non-specific immune responses in an invertebrate species evidenced by a comparative de novo sequencing study[END_REF]. Ferritin may affect the cellular balance of reactive oxygen species (ROS) hydrogen peroxide (H2O2) and hydroxyl free radical (OH) thereby regulating oxidative killing of trematode parasites in B. glabrata [START_REF] Lockyer | Early differential gene expression in haemocytes from resistant and susceptible Biomphalaria glabrata strains in response to Schistosoma mansoni[END_REF]. In vertebrates and invertebrates, various nitric oxide synthase enzymes catalyze production of highly reactive nitric oxide (NO) for immune defense to effect killing of pathogens [START_REF] Nathan | Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens[END_REF][START_REF] Wright | Regulation of nitric oxide production in snail (Lymnaea stagnalis) defence cells: a role for PKC and ERK signalling pathways[END_REF]. In Drosophila melanogaster, NO mediates signal transduction to activate a defense response to Gramnegative bacteria [START_REF] Foley | Nitric oxide contributes to induction of innate immune responses to gram-negative bacteria in Drosophila[END_REF]. Particularly in snail-trematode interactions, killing of S. mansoni by B. glabrata hemocytes involves significant production of NO and H2O2 (Hahn et al., 2001a(Hahn et al., , 2001b)).

Resistant P. columella may benefit from higher constitutive antioxidant potential, as afforded by catalase and SOD, prior to parasite infection because up-regulation of catalase is essential to maintain redox balance and to resist bacterial infection in the clam Meretrix meretrix [START_REF] Wang | The role of catalase in the immune response to oxidative stress and pathogen challenge in the clam Meretrix meretrix[END_REF]. Additionally, high constitutive levels of Cu/Zn SOD are positively related to anti-parasite resistance in B. glabrata snails as this enzyme helps to focus the production of oxygen reactive species from superoxide radical toward H2O2, which is more effective at killing trematode larvae [START_REF] Goodall | Constitutive differences in Cu/Zn superoxide dismutase mRNA levels and activity in hemocytes of Biomphalaria glabrata (Mollusca) that are either susceptible or resistant to Schistosoma mansoni (Trematoda)[END_REF][START_REF] Lockyer | Early differential gene expression in haemocytes from resistant and susceptible Biomphalaria glabrata strains in response to Schistosoma mansoni[END_REF].

The comparative proteomic analysis on products from the albumen gland, showed an overall higher abundance in resistant P. columella of two immune-effectors: LBP/BPI and G-type lysozyme. The G-type lysozymes are lytic proteins with potent activities against pathogens, mainly bacteria. These lysozymes are ubiquitously distributed across animal phylogeny, and have also been reported from several species of molluscs where they can be induced to function as immune effectors [START_REF] Bathige | Evidences for the involvement of an invertebrate goose-type lysozyme in disk abalone immunity: cloning, expression analysis and antimicrobial activity[END_REF][START_REF] Guo | Identification and characterization of a goose-type lysozyme from sewage snail Physa acuta[END_REF][START_REF] Zhang | Three goose-type lysozymes in the gastropod Oncomelania hupensis: cDNA sequences and lytic activity of recombinant proteins[END_REF]. A significant increase of the expression of three Gtype lysozymes was observed in S. japonicum-infected O. hupensis snails and suggests involvement in the defense against trematode parasites also [START_REF] Zhang | Three goose-type lysozymes in the gastropod Oncomelania hupensis: cDNA sequences and lytic activity of recombinant proteins[END_REF].

For the resistant strain of P. columella, the expression of LBP/BPI is also of interest as this protein has been associated with snail parental investment in immune protection of offspring [START_REF] Baron | Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections[END_REF]. Particularly, the albumen gland of B. glabrata produces an isoform of LBP/BPI that is contributed to the perivitelline fluid of snail eggs [START_REF] Hathaway | Identification of protein components of egg masses indicates parental investment in immunoprotection of offspring by Biomphalaria glabrata (Gastropoda, Mollusca)[END_REF] and provides protection against oomycete infections [START_REF] Baron | Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections[END_REF]. Thus, elevated levels of both immune effectors, Gtype lysozyme and LBP/BPI if contributed by the albumen gland of P. columella to the perivitelline fluid, may provide enhanced immune protection for snail embryos that develop inside eggs to potentially compensate for the limited reproductive outputs observed from resistant phenotype snails (Gutiérrez et al., 2002, Alba et al. Under review).

Conclusions

This review of the complex interaction of P. columella with its trematode parasite F. hepatica, focused on the phenotypical, genetical and ecological features of the resistant snail phenotype that occurs in Cuban field populations, underscores the value of in-depth "omics" to reveal the underlying immunobiology. Accordingly, this paper also presents for the first time, a transcriptome of P. columella and, by combining different comparative omic analyses of resistant and susceptible snails, we characterized the molecular baseline of the constitutive biological profile behind the resistance to F. hepatica in naïve P. columella prior to infection.

Similar patterns observed from resistant snails at both the transcriptomic and the proteomic levels, from whole snails and albumen glands, respectively converged to reveal increased abundance of biological processes/transcripts/proteins involved in immune and stress responses and underrepresentation of functions/molecules involved in protein synthesis, that is interpreted to indicate an overall allocation of resources towards survival and self-maintenance in these snails. Specifically, the enhanced immune and stress responsiveness found at the molecular level offer a link between parasite resistance in P. columella and the increased tolerance observed in resistant populations to stress due to low pH and TH variation in the environment. The dedication of metabolic effort to immune and stress response, is indicated to be in detriment of protein synthesis/folding that, especially significant in case of the albumen gland, may amount to reproductive trade-offs and may explain, at least in part, the suggested fitness cost of heightened immune function in snail from Cuban populations of P. columella that are consistently resistant to F. hepatica infection.

However, characterization of differences between naïve resistant and susceptible P. columella snails is only a first step toward the elucidation of the molecular processes and specific mechanisms involved in the resistance to F. hepatica. Analyses of the immunobiological response following parasitic exposure is warranted, progressing to detailed investigation of the role of P. columella hemocytes in response to F. hepatica infection.

Funding sources

Experiments from this study have been financially supported by ANR JCJC INVIMORY (number ANR 13-JSV7-0009) from the French National Research Agency (ANR). AA was financed by the French Embassy in Cuba 2D-electrophoretic profiles of the albumen gland of Pseudosuccinea columella resistant (gel on the left) and susceptible (gel on the right) to Fasciola hepatica infection. Spots identified only in the resistant or susceptible strains are indicated in red and green, respectively. Spots with significant fourfold difference between the two strains are indicated in blue, only on the gel corresponding to the strain in which they are overrepresented. Next to each spot is written the four-digit identification code used in Table 1 and Supplementary File 3. The white rectangle within the gels represents an area of highly abundant proteins with several isoforms left aside of the differential analysis as its complexity impede the quantitative analysis and proper spot and protein identification. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Table 1

Characterization by 2D electrophoresis/Mass Spectrometry of components that differ qualitatively (underlined spot #) or quantitatively between proteomic profiles of albumen gland from resistant and susceptible Pseudosuccinea columella strains.

Figure 1 .

 1 Figure 1. Mantle pigmentation pattern of susceptible (A: Aurora, B: Negrines) and resistant (C: Babiney, D: La Coca, E: El Azufre, F: La Palma) Pseudosuccinea columella populations to Fasciola hepatica infection. The arrows indicate the characteristic band of small sharp spots in the mid-region of the mantle of resistant snails. (Photos: Laboratory of Malacology, Institute "Pedro Kourí" of Tropical Medicine).

Figure 2 .

 2 Figure 2. A) Enriched biological process and B) molecular functions of Pseudosuccinea columella transcriptome that are involved (overrepresented; in red) in the resistant phenotype. C) Enriched biological process and molecular functions of P. columella transcriptome particularly underrepresented (in blue) in the resistant phenotype. Each bar represents the percentage of unisequences found in the test data (over or under-represented transcripts in resistant P. columella) according to their GO terms. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

  Figure 4.2D-electrophoretic profiles of the albumen gland of Pseudosuccinea columella resistant (gel on the left) and susceptible (gel on the right) to Fasciola hepatica infection. Spots identified only in the resistant or susceptible strains are indicated in red and green, respectively. Spots with significant fourfold difference between the two strains are indicated in blue, only on the gel corresponding to the strain in which they are overrepresented. Next to each spot is written the four-digit identification code used in Table1and Supplementary File 3. The white rectangle within the gels represents an area of highly abundant proteins with several isoforms left aside of the differential analysis as its complexity impede the quantitative analysis and proper spot and protein identification. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Acknowledgments

This study is set within the framework of the "Laboratoires d'Excellences (LABEX)" TULIP (ANR-10-LABX-41). Authors acknowledge Dr. Silvain Pinaud and Dr. Ricardo Lebrón for valuable comments on the transcriptomic analyses. The authors would like to thank the insightful comments and suggestions made by the anonymous reviewers and by the editor that greatly helped in improving the manuscript.

We are in debt to Dr. Coen Adema for the excellent editorial work.

Author's contributions AA, GT, CC, JS, AAV, BG designed and performed the experiments and analysis, and participated in the reviewing process. AA, AAV, JS participated in snail breeding, and sample recollection. AA, GT: conducted the proteomic experiment and analyses. AA, CC, BG conducted the transcriptomic experiment and analyses. AA, GT, BG drafted the manuscript. All authors read and approved the final version of the manuscript.

All data generated or analyzed during this study are included in this article [and its supplementary information files]. All RNAseq data is available online under the following accession numbers: NCBI-SRA submission number: SUB5947916; BioProject: PRJNA555222: Pseudosuccinea columella RNA sequencing; BioSample: SAMN12305757: Pseudosuccinea columella RNAseq (TaxID: 31228).

Full and simplified transcriptomes of P. columella snail are also available at: http://ihpe.univperp.fr/acces-aux-donnees/