Annia Alba
email: annia.alba@gmail.com

Antonio A Vázquez

Jorge Sánchez

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

Immunological Resistance of Pseudosuccinea columella Snails From Cuba to Fasciola hepatica (Trematoda) Infection: What We Know and Where We Go on

Keywords: host-parasite interaction, snail immunity, cost of resistance, selection of resistance to parasitism, ecological immunology, evolution

whether they are published or not. The documents may come

Introduction

The effects of parasitism on host's populations are significant; (i) from exerting densitydependent regulation, (ii) actively contributing to the diversity patterns of host's populations, (iii) affecting their genetic structure through selection, and possibly, (iv) the maintenance of sexual reproduction, to (v) leading in many cases, to co-speciation through host-parasite interactions [START_REF] Little | Genetic variation in a host-parasite association: potential for coevolution and frequency-dependent selection[END_REF]. Concomitantly, the study of host-parasite interactions is particularly essential for the comprehension of the epidemiology and transmission of infectious diseases as there is a reciprocal natural selection between host resistance and parasite infectivity that impacts not only the spatio-temporal dynamics of parasite circulation but also the genetic structure of both entities [START_REF] Hawley | Disease ecology meets ecological immunology: understanding the links between organismal immunity and infection dynamics in natural populations[END_REF]. In this sense, such interplay also contributes to shaping the host's defense system, with the latter evolving under the influence of two different but equally important selective pressures: to fight back, in a regulated and effective manner, the plethora of infective or harmful agents while remaining tolerant to host self (including microbiota).

As parasites continuously evolve to improve their fitness for infecting, surviving, and to be transmitted while thriving at the expenses of host resources, a reduction in fitness is expected to occur in parasitized hosts compared to non-parasitized, due to the inherent mechanical, energetic and/or physiological damage exerted by the parasite. This could result in a significant selective pressure as, in turn, hosts must adapt to parasite changes to resist or to tolerate the aggression in an evolutionary "arms race" [START_REF] Hurtrez-Boussès | Dynamics of host-parasite interactions: the example of population biology of the liver fluke (Fasciola hepatica)[END_REF]. Nonetheless, examples of host resistance in nature are not plentiful, as these very rare genotypes are usually less abundant due to a fitness cost of such resistance in the absence of parasitism [START_REF] Lively | Parasite adaptation to locally common host genotypes[END_REF]. Notwithstanding, a few different types of selection of resistance have been suggested from theoretical studies on the evolution of resistance to parasites (see [START_REF] Duffy | Ecological feedbacks and the evolution of resistance[END_REF] for review). Briefly, there is the well-known i) frequency-dependent selection related to co-evolution of hosts and parasite under the Red Queen principle in which the parasite "tracks" the common hosts genotypes, ultimately reducing their fitness and favouring rare genotypes in a time-lagged parasite-driven genetic selection in host populations. Other theories include ii) the directional selection for increased resistance or iii) susceptibility, and iv) the stabilizing or v) disruptive selections, all of which are tightly related to the extent of the selective pressure exerted by the parasite and/or to the degree of the associated cost of resistance. The type of selection that occur varies between particular localities or years and it is postulated that such variation is strongly related to the nature, strength, and shape of the trade-offs associated with resistance, which are highly influenced by ecological factors [START_REF] Duffy | Ecological feedbacks and the evolution of resistance[END_REF].

The acknowledgement of the impact of environmental variables (e.g. climate, seasonality, other infections, etc.) on host-parasite interactions has been on the rise with several studies accounting for their direct influence on host susceptibility and pathogen dynamics, as environmental changes can alter the encounter and compatibility filters, the immune competence of the host and the immunobiological interplay of the system, and the final outcome of infection (see [START_REF] Budria | How does human-induced environmental change influence hostparasite interactions[END_REF][START_REF] Lafferty | How environmental stress affects the impacts of parasites[END_REF][START_REF] Wolinska | Environment can alter selection in host-parasite interactions[END_REF] for review). Furthermore, the indelible link between ecological interactions and evolutionary changes through space and time in host and parasite populations (and their interplay) determines the need for integrative approaches across multiple scales of biological organization [START_REF] Schmid-Hempel | Evolutionary Parasitology: The integrated study of infections, immunology, ecology and genetics[END_REF]. This would help to comprehend evolutionary dynamics of disease transmission, to identify general processes and principles within host-parasites interactions that transcend taxonomic boundaries [START_REF] Hawley | Disease ecology meets ecological immunology: understanding the links between organismal immunity and infection dynamics in natural populations[END_REF], and to attempt the understanding of spatio-temporal variation in ecological factors and their influence on parasite-mediated selection through associated trade-offs for resistance [START_REF] Duffy | Ecological feedbacks and the evolution of resistance[END_REF].

Within host-parasite systems, snail-trematode models are significant for comparative immunology, evolutionary biology and health sciences [START_REF] Loker | Gastropod immunobiology[END_REF]. The class Trematoda (Platyhelminthes) groups many parasites highly relevant to public and veterinary health and they present an almost strict dependence upon molluscs, particularly snails acting as intermediate hosts. As a result, snails need to cope with the deleterious effects of infection in an ever-changing environment. As a very interesting model, the interaction of the snail Pseudosuccinea columella with the trematode Fasciola hepatica has aroused a multi-approach interest integrating disciplines, from classical malacology and parasitology to combined immunological, ecological and evolutionary perspectives. This snail-trematode system stands out by presenting two different phenotypes within the same species P. columella when infection by F. hepatica occurs: some populations have susceptible individuals (with a varying degree of compatibility among populations) while others are completely resistant to the parasite. There are studies that show the worldwide distribution of susceptible populations [START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at the worldwide scale[END_REF] usually displaying variations in compatibility traits (see [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] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF] for discussion). Such variation, dependant on the genotypes of the infecting parasites, reveals a co-evolution towards a certain degree of genetic matching between the host and the parasite known as polymorphism of compatibility (Thèron andCoustau, 2005, Thèron et al., 2014). However, resistant populations have been only reported until now in specific natural field sites within the Caribbean island of Cuba and whenever tested, always succeed in fighting back the infection regardless of the F. hepatica isolate, the parasitic dose, or even the infection scheme used (e.g. [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] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF], Alba et al., 2018). Immunological and molecular studies reflect a constitutively enhanced immune competence of resistant compared to susceptible populations [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF], Alba et al., 2019a), although such selection for resistance is thought to carry a cost in fitness in the absence of parasites; reproductive constraints have been associated to resistant P. columella (Alba et al., 2019a[START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in 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 this sense, distinctive ecological patterns have been related to each phenotype; in particular, resistant P. columella always occurs in relatively soft/acid waters with low malacological diversity [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF].

Herein, we will briefly present this host-parasite system by reviewing the current knowledge and advances made on the topic of P. columella resistance from different biological perspectives including phenotypic, molecular, immunological and ecological standpoints.

Afterwards, we will discuss the possible origins of the resistant phenotype based on the preliminary evidences and on theoretical understanding of parasite and environmental-driven selection. Lastly, as much remains to be studied for a proper understanding of the mechanistic and evolutionary bases featuring the resistant/susceptible phenotypes, clues into some future experimental perspectives will be briefly stated. The advances revised herein are beginning to unravel mechanisms of anti-parasite innate defense responses and their evolutionary bases, and can facilitate the development of prospective approaches towards practical applications of P. columella resistance.

Resistance of P. columella to F. hepatica in Cuba: what we know

Cuba, the biggest archipelago of the Caribbean basin, is mainly plains and floodable lowlands under a tropical regime of warm temperatures and frequent rainfall throughout the year, all of which favor the occurrence of the two lymnaeid snails Galba cubensis and P. columella [START_REF] Vázquez | Distribución y preferencia de hábitats de moluscos hospederos intermediarios de Fasciola hepatica en Cuba[END_REF]. Both species act as intermediate hosts of a number of parasites, including the medically and veterinary relevant trematode F. hepatica [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][START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[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], the main etiological agent of the (re-)emergent significant zoonosis known as fasciolosis (see [START_REF] Alba | Towards the comprehension of fasciolosis re-emergence: an integrative overview[END_REF]). In this sense, elevated rates of transmission of F. hepatica in nature (mainly related to human activities such as cattle husbandry) are believed to occur all year-round [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., 2016) supported by high prevalence of infection in both lymnaeid snail species reported from field [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][START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF][START_REF] Vázquez | Natural prevalence in Cuban populations of the lymnaeid snail Galba cubensis infected with the liver fluke Fasciola hepatica: small values do matter[END_REF] and laboratory studies (e.g. [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] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF]). Although ecological and parasitological studies point at G. cubensis as the main intermediate host of F. hepatica in Cuba and in the Caribbean [START_REF] Alba | Desarrollo de ensayos inmunoenzimático y molecular para la detección de Fasciola hepatica (Trematoda: Digenea) en moluscos hospederos: evaluación del sistema FasciMol-ELISA como herramienta para la vigilancia epidemiológica[END_REF][START_REF] Vázquez | Distribución y preferencia de hábitats de moluscos hospederos intermediarios de Fasciola hepatica en Cuba[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], Vázquez et al., 2019), P. columella is also a worldwide-recognized host of the parasite [START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at the worldwide scale[END_REF]. In Cuba, P. columella presents a wide distribution in the western and central regions of the country [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF]. Moreover, high infection rates of P. columella by F. hepatica from 1% to 10% [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[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], and even up to 100 % prevalence [START_REF] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF] have been recorded respectively from the field and from experimental studies in Cuba.

However, one of the most interesting facts related to P. columella in Cuba is the occurrence of, at least six field populations that are naturally resistant to F. hepatica infection. The six populations occur in the localities of La Playita, Loma Candelaria, La Palma, El Azufre, La Coca, and Babiney, with the latter being the only one found in central Cuba (see [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF] for details on their distribution). A distinctive morphological feature consisting of a belt-like shape of small, highly abundant sharp-whitish spots in the central region of the mantle with wider and sparser spots in the upper and lowers sides has been associated to these populations (see (Alba et al., 2019a, Gutiérrez et al., 2003b), and Figure 1). All P. columella snails displaying such phenotypic markers have never been found infected with F. hepatica, either in nature or after experimental challenges, regardless of the isolates of the parasite used, the parasite dose or the infection scheme [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]. In this sense, histological sections of exposed resistant P. columella showed that the penetration of F. hepatica miracidium into the snail is rapidly followed by an effective encapsulation of the parasite larvae by the host's hemocytes (circulating immune cells of snails), observed at 24 h post-exposure (Gutiérrez et al., 2003b). This powerful immunological reaction is believed to be at the base of the resistance of P. columella to F. hepatica [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF], Gutiérrez et al., 2003b).

Although resistant and susceptible P. columella belong to the same species accordingly to significant anatomical, conchiological and genetic data (Alba et al., 2019a, Gutiérrez et al., 2003a), nuclear polymorphic genetic markers and mitochondrial haplotypes cluster resistant populations in a different clade from susceptible snails, with little within-group polymorphism [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in 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] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at the worldwide scale[END_REF]. This segregation implies a certain genetic determinism of resistant P. columella and opens to the possibility that such phenomenon was selected from or has derived in a different genetic pool of the majority of susceptible P. columella populations worldwide [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF][START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at the worldwide scale[END_REF]. Furthermore, resistant populations, particularly those from La Coca and Babiney, showed an overall higher allelic richness and higher genotypic diversity than susceptible populations, and they not only significantly differed from susceptible populations but also between themselves, possibly as a result of different introductions between western and central regions of Cuba and to their in situ evolution [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF].

Comparative molecular and immunobiological studies between P. columella phenotypes: evidences of increased immune competence towards F. hepatica associated with the resistant phenotype

For two decades, several efforts have been made to challenge the resistant phenotype by increasing the odds for F. hepatica success. In this sense, resistant P. columella snails have been exposed to different sympatric and allopatric F. hepatica from Cuba (e.g. [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] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF], which are usually genetically diversified (see [START_REF] Vázquez | Genetic and infective diversity of the common liver fluke Fasciola hepatica (Trematoda: Digenea) from Cuba[END_REF] for details). In addition, they have been also confronted against isolates of F. hepatica from different countries (Dominican Republic -narrow geographic scale and France -large geographic scale), trickle infections (3 exposures every 3 days) and single infections with increasing parasite loads [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]. Although these attempts aimed at increasing the probability of encounter of compatible genotypic host-parasite individual combinations and at potentially decreasing the effectivity of the immune response of the host (such effects have been observed in susceptible populations used as control), they have all failed in rendering phenotypically resistant snails infected. Such stability and universality (at least at the above mentioned scale) of this phenotype paired to the potent immune response associated to it [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF], Gutiérrez et al., 2003b), point at the occurrence of an immunological resistance that is not restricted by specific interactions of matching genotypes between the host and the parasite. It is, therefore, rather a phenomenon of immunological resistance than one of incompatibility and that, noteworthy, has been selected in nature (Alba et al., 2019a, Gutiérrez et al., 2003b). We have postulated that a major factor/function associated to a single locus or a few loci is present or highly abundant in the genetic clade of resistant P. columella snails. Such feature is believed to be involved proximately or ultimately, in the immunobiological interaction with F. hepatica, interfering with biologically relevant functions of the parasite coded by a single locus or a few loci highly conserved [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF]. Contrastingly, given the polymorphism of compatibility resulted from exposing different susceptible snails to different parasite isolates (see [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] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF] for discussion), it was speculated that the major factor highly present in resistant snails is absent/scarce in susceptible P. columella and, consequently, it is the interplay among several other factors/functions between the susceptible host and the parasite that dictates the final outcome and the intensity of the infection, possibly preferentially following a matching allele model [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF].

In this sense, the comprehension of the molecular and mechanistic bases that mediate P. columella resistance to F. hepatica is pivotal for comparative immunology and evolutionary biology. The rapid orchestration of the protective cellular immune response against F. hepatica in the resistant phenotype during the first 24 h post-infection (Gutiérrez et al., 2003b) supports the notion of the occurrence of at least some constitutive elements participating in the resistance (Alba et al., 2019a). In fact, hemocytes from naive (nonexposed) resistant snails significantly associate to higher encapsulation activity of F. hepatica sporocysts in vitro, compared to immune cells from naive susceptible snails [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF]. With all this in mind, the whole-snail transcriptome of P. columella and the differential proteome of the albumen gland (organ involved in both reproduction and immune response) prior infection (naïve conditions) were analyzed to compare the constitutive "molecular base line" between resistant and susceptible phenotypes (Alba et al., 2019a).

From this study, significant enrichments of five functions/biological processes in the resistant phenotype were recorded, among which the immune/stress response was particularly noted. Such enrichment in this biological functions was supported by the significant abundance of different transcripts identified as orthologues of recognized immune molecules involved in the recognition of pathogens, in immune regulation, and defense activation/response in association to resistant P. columella. In this sense, several types of lectins and pattern recognition receptors, CD109, cytokines and cytokine related 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 signalling/regulatory transcripts (e.g. Toll-like receptors (TLR), protein C kinase (PKC), members of the superfamily of tumoral necrosis factor (TNF) receptors, interferon regulatory factors (IRF) and molecules with leucine rich repeats), and enzymatic antioxidants and molecular effectors such as ferritine, nitrite oxide synthase, catalase and superoxide dismutase where found overexpressed in resistant versus susceptible snails (Alba et al., 2019a). Orthologues of these transcripts have been recognized as relevant for the immune response in other mollusk species such as Lymnaea stagnalis and Biomphalaria glabrata, and particularly, in their defense against trematodes (e.g. [START_REF] Baeza-Garcia | Involvement of the cytokine MIF in the snail host immune response to the parasite Schistosoma mansoni[END_REF][START_REF] Hahn | Involvement of nitric oxide in killing of Schistosoma mansoni sporocysts by hemocytes from resistant 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] Lockyer | Early differential gene expression in haemocytes from resistant and susceptible Biomphalaria glabrata strains in response to Schistosoma mansoni[END_REF], Pila et al., 2016b[START_REF] Wu | Proteomic analysis of Biomphalaria glabrata plasma proteins with binding affinity to those expressed by early developing larval Schistosoma mansoni[END_REF]. In addition, other significant immune effectors, i.e. a G-type lysozyme and a lipopolysaccharide-binding protein/bactericidal permeability-increasing protein (LBP/BPI) [START_REF] Baron | Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections[END_REF][START_REF] Zhang | Three goose-type lysozymes in the gastropod Oncomelania hupensis: cDNA sequences and lytic activity of recombinant proteins[END_REF], were also revealed from the analysis of the albumen gland to be only present in the proteome of resistant snails (Alba et al., 2019a). The latter made patent, at the molecular level, the significant and constitutive allocation of resources towards self-maintenance, which possibly occur at the expenses of other competing biological functions (e.g. reproduction; [START_REF] Stearns | The Evolution of Life Histories[END_REF]) and thus, implying possible trade-offs associated to resistance.

In terms of P. columella -F. hepatica interaction, apparently, mannosylated residues in the surface of F. hepatica sporocysts and mannose recognition factors have central roles in the molecular interplay between the parasitic larvae and the snail hemocytes. Indeed, a marked inhibition of parasite encapsulation by P. columella hemocytes was observed when the cells were pre-incubated with mannose, irrespective of the phenotype [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF]. Among the previously mentioned overrepresented molecules associated with resistant P. columella, the high abundance of different types of lectins and in particular, those involved in mannose recognition, e.g. mannose-binding protein C, C-type mannose receptor 2, mannose binding protein, paired with the overexpression of immune-related signalling molecules (Alba et al., 2019a) might be fundamental in driving a protective response in the resistant phenotype that begins by the proper recognition of F. hepatica. At the functional level, qualitative and quantitative differences in the surface of hemocytes between phenotypes can be presumed from the different degrees of inhibition of the encapsulation of F. hepatica by P. columella hemocytes recorded when the cells were pre-incubated with a variety of carbohydrates (i.e. glucose, galactose, fructose; [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF]). Other functional differences were observed when different aspects of the immune response were studied and compared between P. columella phenotypes following experimental infection. A significant increase of the number of circulating hemocytes, of the proliferative activity of blast-like hemocytes and in the adherence and spreading capacity of large hemocytes were verified to occur in resistant snails, at 24 h post-exposure to F. hepatica. Notably, the highest in vitro encapsulation activity, resulting in the total formation of a hemocyte capsule around F. hepatica sporocyst was recorded only in exposed resistant snails, at 24 h post-exposure [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF]. Additionally, a rapid increase of the expression of the cytokine granuline was recorded at 6h post-exposure only in resistant snails [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF]. This cytokine participates in the proliferation, activation and differentiation of hemocytes in trematode-challenged snails and increased levels have been associated to a decrease of the susceptibility to the infection in B. glabrata -Schistosoma mansoni system (Pila et al., 2016a).

Therefore, not only do resistant snails present a more competent molecular and functional base line prior infection, but they also respond more rapidly and efficiently to the parasite following infection (Alba et al., 2019a[START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF]. These features endorse the potent immune phenotype that develops in these snails towards F. hepatica and indicate that constitutive and inducible elements share roles in the orchestration and amplification of the protective immune response that occurs in resistant P. columella [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF].

Contrastingly, exposure to F. hepatica of susceptible snails rendered a less competent immune phenotype not only compared to resistant snails but also to their naïve conditions. No significant increase, neither of the total hemocytes counts nor of blast-like cells proliferation, were observed in exposed-susceptible snails. In addition, large hemocytes displaying less and/or shorter pseudopods without clear tendency to cohesion or aggregation at 24 h postexposure compared to naïve conditions were noted, which possibly relates to the diminishment of the encapsulation capacity of F. hepatica larvae also recorded at that time point. Furthermore, a significant decrease of granulin expression at 12h and 24h time points, below constitutive levels, observed in the susceptible phenotype accounted for an almost 3-fold differential increased expression in resistant compared to susceptible snails at 24h postexposure [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF]. From these results, we suggested that infection by F. hepatica involves immunomodulatory/immunosuppressive parasite-driven effects that are rapidly put in place and that only affect the susceptible phenotype, possibly resulting in infection when compatible combinations take place. It has been hypothesized that the constitutive allocation of resources to defence observed in resistant snails contributes to speedily impair such parasitemediated immunomodulation or that F. hepatica immunosuppressive factors are inefficient against resistant snails and, concomitantly, trigger the powerful protective response that develops following F. hepatica penetration (see [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF] for discussion).

Selection for resistance is often associated with fitness costs, thus understanding how resistant phenotype could maintained in the field is an intricate question. To answer this question it is important to consider the environmental conditions in which host and parasite species evolved. This is particularly true for resistant P. columella that always occurs in relatively soft/acid waters with low malacological diversity. The links between resistance, fitness cost and environmental factors need thus to be investigated in the P. columella / F. hepatica interaction.

Ecological patterns associated with the resistant phenotype: evidences of a cost for resistance and environmental abiotic factor selective pressures

Taking into account the heavy investment on immune or stress response described in P. columella (see section 2.1), particularly in naïve conditions, one could ask if is an ecological cost of P. columella resistance to F. hepatica in the absence of parasitism?

Results from [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF] indicate that from all the malacological records of P. columella in Cuba, the majority (more than 90% of all field records) belongs to the susceptible phenotype. This contrasts with the supposition that resistant populations are the result of more ancient introductions in the island given its high genetic diversity in relation to the mostly monomorphic nature of susceptible P. columella snails from Cuba. This is particularly interesting knowing the diminished competitive potential of resistant snails compared to susceptible snails, resistance seems to impair their apparent capacity to invade and successfully colonize new habitats. In this sense, the analysis of the ecological factors occurring in the different sites where each P. columella phenotype exists has rendered a peculiar association: resistant P. columella populations were always present in sites of slightly acidic (pH=6.2+0.12), softer water (total hardness, TH=6.3+1.03°d) and lower malacological diversity (3.2+1.02 freshwater snail richness; [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF]). Extensive ecological studies performed state that most freshwater sites in Cuba are featured by higher pH/TH values (ranging from 7-8.5 and 12-18°d; [START_REF] Perera | Écologie des mollusques d'eau douce d'intérêt médical et vétérinaire a Cuba[END_REF]), and in fact, resistant populations represent only the 1.2% of all the registered accounts of freshwater snail populations in Cuba [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF].

To clarify the significance of pH/TH on the occurrence of resistance and susceptible P. columella, life-history traits experiments were performed in the laboratory simulating the differential values recorded in the field in association with each phenotype (i.e. 5.9/4°d ó 7.8/14°d; [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF]). According to this study, survival and fertility traits of P. columella snails are negatively affected by low pH/TH irrespective of the phenotype whereas, overall, all populations performed better at common pH/TH values (similar to those corresponding to the finding of susceptible populations in the field). Nonetheless, resistant populations showed a diminished reproductive potential featured by a lower fecundity rate and late reproductive peaks (La Coca population) or by delayed egg hatching (La Palma population) when compared to susceptible P. columella, regardless of the experimental setting [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF]. Previous studies also reported lower fecundity rates for resistant (La Palma population) compared to susceptible P. columella [START_REF] Gutiérrez | Fasciola hepatica (Trematoda: Digenea): its effect on the life history traits of Pseudosuccinea columella (Gastropoda: Lymnaeidae), an uncommon interaction[END_REF]. All these results suggest a possible fitness and physiological cost associated with the resistant phenotype of P. columella [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] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF]. Interestingly, resistant snails showed higher tolerance to the low pH/TH setting than susceptible snails. Resistant snails presented higher survival, life expectancy at birth and proportion of viable eggs compared to susceptible snails [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF]. These results suggest that the restricted distribution of the resistant phenotype in low pH/TH water is due to the conjunction of their higher tolerance to these stringent environmental conditions and the compensation of their diminished reproductive and competitive abilities in sites of low malacological diversity [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF]. It has been reported, under experimental conditions, that the presence of susceptible P. columella when rearing resistant snails results in a decrease of the net reproductive rates of the latter. Contrastingly, susceptible snails increase both growth and net reproductive rates when paired with resistant snails (Gutiérrez et al., 2005b), which could be related to an unbalanced competition in favor of susceptible snails. In nature, a temporal invasion of a traditionally susceptible P. columella -occurring site by a nearby resistant population coincide with the decrease of pH/TH values from 7.5 to 6.5 [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF]. Furthermore, ecological follow-up studies of temporal dynamics on El Azufre and La Coca (sites of low pH/TH) have shown that resistant P. columella occurs always as the more stable population compared to the other mollusk species, even compared to the acknowledged highly invasive gastropods Tarebia granifera and Physa acuta (see (Gutiérrez et al., 2005a[START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF]). Both species are widely distributed in Cuba, and particularly T. granifera, has been pointed as strong competitor for pulmonates including lymnaeids.

It is significant to point out that results from the molecular studies are in harmony with what have been observed from the ecological approaches. The molecular base line of resistant P. columella is characterized by a significant allocation of resources to the constitutive production of molecules involved in immune defense and stress responses, which could not only endorse the enhanced capacity to react to F. hepatica infection but also to abiotic stressors such as particular environmental conditions (Alba et al., 2019a). In this sense, and concordantly with the higher tolerance displayed for pH/TH variations, several transcripts, particularly involved in the regulation of osmotic and pH variations of the internal milieu [START_REF] Freitas | Biochemical alterations induced in Hediste diversicolor under seawater acidification conditions[END_REF][START_REF] Mattews | Acid-base balance and nitrogen excretion in invertebrates: mechanisms and strategies in various invertebrate groups with considerations of challenges caused by ocean acidification[END_REF], e.g. several carbonic anhydrase isoforms, and major ion transporters such as Na + /K + -ATPase, H + -ATPase, Na + /H + -exchanger, are constitutively overexpressed in resistant compared to susceptible snails (see (Alba et al., 2019a)). This constitutive over-expression of genes involved in adaptation to acidic water in the resistant phenotype may specifically tradeoff against reproduction. Concomitantly , resistant snails trigger a powerful protective immune cellular response involving the production of immune effectors following F. hepatica penetration [START_REF] Alba | The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: hemocytes in the spotlight[END_REF], resulting also in a potential reproduction trade-off. This is particularly evidenced by the comparative proteomic approach conducted on the albumen gland. Albumen gland guarantees the nourishment of embryos through the production of different elements of the perivitelline fluid (Duncan, 1975, Geraerts and[START_REF] Geraerts | Freshwater snails (Basommatophora)[END_REF], but is also involved in production of immune effectors. Thus, the metabolic rearrangements favouring the production of particular immune effectors, regulatory and catabolic enzymes in detriment of overall protein synthesis in albumen gland could be at the base of the reproductive constraints associated to the resistant phenotype (Alba et al., 2019a).

Possible influence of parasite vs. environmental factors: discussing the selection of resistance

As previously mentioned, resistant P. columella populations could tolerate slightly acid and soft waters better than susceptible snails and they experienced certain reproduction constraints compared to susceptible populations [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in 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]. Interestingly, both, a protective immune response towards F. hepatica and an enhanced tolerance to pH/TH variations, involved higher molecular and phenotypical fitness to respond to stressors (biotic or abiotic), whereas the impairment of reproductive traits is likely a consequence of energetic trade-offs among potentially competing physiological processes (i.e. self-maintenance/survival vs. reproduction; [START_REF] Stearns | The Evolution of Life Histories[END_REF]). All these could explain the limited distribution of resistant P. columella in the field to localities where being more tolerant to variations of water acidity/hardness or more immunologically resistant could be considered as an advantage (i.e., sites with low pH/TH or high F. hepatica pressure) thus, compensating for their low reproductive or competitive capacities.

However, the question of the origins of the primary feature of resistant snails (i.e. the immunological resistance to F. hepatica) and its association with the higher tolerance to pH/TH variations remain. Noteworthy, both the immune response and acid-base/osmotic regulation, are physiologically linked through several molecular functions, effectors and signalling 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]. Changes on environmental and internal pH could lead to an improved immunological condition through an increase of different defence-related traits as observed in other invertebrates, e.g. increase haematopoiesis [START_REF] Meseck | Ocean acidification affects hemocyte physiology in the Tanner Crab (Chionoecetes bairdi)[END_REF], phagocytic capacities of the hemocytes [START_REF] Matozzo | First evidence of immunomodulation in bivalves under seawater acidification and increased temperature[END_REF][START_REF] Meseck | Ocean acidification affects hemocyte physiology in the Tanner Crab (Chionoecetes bairdi)[END_REF], and acid-balance and anti-oxidative regulatory potential [START_REF] Freitas | Biochemical alterations induced in Hediste diversicolor under seawater acidification conditions[END_REF]. The boosting of such features with pivotal roles in processes such as immune surveillance and immune response (see [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] Larson | Resistance of Biomphalaria glabrata 13-16-R1 snails to Schistosoma mansoni PR1 is a function of haemocyte abundance and constitutive levels of specific transcripts in haemocytes[END_REF][START_REF] Connors | Schistosoma mansoni: interleukin-1 increases phagocytosis and superoxide production by hemocytes and decreases output of cercariae in schistosome-susceptible Biomphalaria glabrata[END_REF][START_REF] Nikapitiya | Characterization and expression analysis of EF hand domain-containing calcium-regulatory gene from disk abalone: calcium homeostasis and its role in immunity[END_REF] for some examples), could make the snails more prone to efficiently respond to infectious challenges by being "better prepared" to recognize, phagocyte/encapsulate and kill intruders. On the other hand, an enhanced immune responsiveness may involve a metabolic reconfiguration towards a relative increase of pathways related to energy and metabolite production; e.g. respiratory rate, glycolysis, proteolysis and/or lipolysis [START_REF] Lochmiller | Trade-offs in evolutionary immunology: just what is the cost of immunity?[END_REF]. This could lead to a concomitant increase of protons, reactive oxygen species and CO2 levels in the internal milieu, a biochemical setting that it is, then, coped through an enrichment of other related functions, such as antioxidant enzymes, carbonic anhydrase and certain ion transporters [START_REF] Epstein | Separation of metabolic supply and demand: aerobic glycolysis as a normal physiological response to fluctuating energetic demands in the membrane[END_REF][START_REF] Biller-Takahashi | The immune system is limited by oxidative stress: Dietary selenium promotes optimal antioxidative status and greatest immune defense in pacu Piaractus mesopotamicus[END_REF]. Therefore, the enhanced tolerance to pH/TH variations associated to resistant snails could be a result of such a metabolic reconfiguration as, in aquatic species, the acid-base and osmotic balances are closely linked through the coupled movement of H + and HCO3 -with that of Na + via ion transporters [START_REF] Mattews | Acid-base balance and nitrogen excretion in invertebrates: mechanisms and strategies in various invertebrate groups with considerations of challenges caused by ocean acidification[END_REF]. In this sense, an overexpression of immune-related transcripts/proteins with pro-activating and proinflammatory roles, an enrichment of energetic metabolic process, and of carbonic anhydrase and major ion regulators featured the constitutive molecular base line of the resistant phenotype (Alba et al., 2019a).

Unfortunately, all these experimental and theoretical outlines remain inconclusive and from them, the hypothesis on the selection of the primary feature associated to resistance could go either way: is it the higher pH/TH tolerance a "by-product" resulting from parasite-driven selection of resistance, or is it the resistant phenotype a merely "side effect" of an environmental-driven selection for an increased fitness for compensating osmotic and acidbase variations? An environmental-driven selection could, perhaps, easily explain the broad resistance towards F. hepatica, irrespective of the infective isolate (see [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] Vázquez | Fasciola hepatica in Cuba: compatibility of different isolates with two intermediate intermediate hosts, Galba cubensis and Pseudosuccinea columella[END_REF]), through an overall enhancement of multiple and "universal" immune functions in the snails. However, from such an environmentally-linked immune reactiveness not selected from specific parasitic pressures, it might be also expected for it to remain fairly "constant" and not to strongly develop into a highly competent induced defence following a particular infection, and possibly, for it to be extended to other infectious challenges. Contrastingly, evidences of a potent and significantly different induced immune response following exposure to F. hepatica in terms of hemocyte morphology, proliferation and encapsulation activities, and of granulin expression are reported from resistant but not from susceptible P. columella snails. Contrastingly, resistant and susceptible P. columella were equally sensitive experimental infection by Trichobilharzia sp. from which high infection rates and cercarial outputs were attained (see [START_REF] Alba | Comparative biology of susceptible and naturally-resistant Pseudosuccinea columella snails to Fasciola hepatica infection in Cuba: ecological, molecular and phenotypical aspects[END_REF]). Moreover, it is noteworthy that resistant snails from La Coca have been found naturally infected with rediae, cercariae and metacercariae of Echinostomatidae (author's unpublished data). These results suggest that there is a certain specificity of such "enhanced immune competence" towards F. hepatica that allows to uniquely recognize and to "better" respond to this particular parasitic challenge and thus, possibly fitting better into a parasite-driven selection hypothesis.

However, we have to be careful to not oversimplify the situation, considering exclusively a simple dichotomy, without considering that both selection favoring resistance and selection favoring pH tolerance could be involved in the observed natural resistant phenotype. To present an integrative view of all these results and of the possible influence of both the parasite and the environment, we hypothesized that a parasite-driven selection took place resulting in a phenotype featured by an immunological resistance to F. hepatica and a high tolerance to pH/TH changes that traded-off against reproduction. Frequency dependent selection and/or disruptive selection on resistance associate evolution of resistance through parasite mediatedselection with the occurrence of a fitness cost and of an increase of the genetic diversity of host populations (see (Duffy et al., 2008, Dybdahl and[START_REF] Dybdahl | Host-parasite coevolution: Evidence for rare advantage and time-lagged selection in a natural population[END_REF] for examples), could have occurred in the case of resistant P. columella (see [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in 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][START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at the worldwide scale[END_REF] for genetic diversity and cost of resistance in P. columella)). While it should be kept in mind that evolutionary processes are time-lagged, it might be argued that impaired competitive capacities due to the trade-off against reproduction could have probably already determined a negative selection for the resistant but less ecologically-competitive phenotype with the diminishment of the parasitic pressure that followed the primary selection for resistance. However, it should be considered that resistant P. columella occurred mostly in western Cuba [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF], a region that has been recognized for decades to be one of the most heavily impacted by fasciolosis [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF][START_REF] Kourí | Helmintología humana[END_REF][START_REF] Rojas | Fascioliasis: can Cuba conquer this emerging parasitosis?[END_REF], Vázquez et al., 2016, Alba et al., 2016). In particular, four resistant populations are even located within or in close proximity to localities where specific transmission foci of F. hepatica have been described (i.e. La Palma, El Azufre, La Coca, Loma de Candelaria; see [START_REF] Alba | Desarrollo de ensayos inmunoenzimático y molecular para la detección de Fasciola hepatica (Trematoda: Digenea) en moluscos hospederos: evaluación del sistema FasciMol-ELISA como herramienta para la vigilancia epidemiológica[END_REF], Gutiérrez et al., 2005a[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] Wong | Estudios ecológicos en moluscos de importancia médico-veterinaria en la granja de desarrollo La Coca[END_REF]). Furthermore, high prevalence of infection in lymnaeid snails [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][START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF] and livestock [START_REF] Vázquez | Genetic and infective diversity of the common liver fluke Fasciola hepatica (Trematoda: Digenea) from Cuba[END_REF][START_REF] Brito | Prevalencia, decomisos de hígado y pérdidas económicas por Fasciola hepatica en mataderos bovinos de tres provincias de la región central de Cuba[END_REF], and even human cases and outbreaks [START_REF] Díaz | Comportamiento clínicoterapéutico de Fasciola hepatica en una serie de 87 pacientes[END_REF][START_REF] Rojas | Fascioliasis: can Cuba conquer this emerging parasitosis?[END_REF] have been historically reported in central and western Cuba, all of which presume high odds for host -parasite encounters all year-round. Snail-trematode encountering rate has proved to be significant in the field, even in sites showing low prevalence of patent infections, by the use for example of sentinel snails for Bulinus globosus/Schistosoma haematobium interaction [START_REF] Allan | Use of sentinel snails for the detection of Schistosoma haematobium transmission on Zanzibar and observations on transmission patterns[END_REF]. Concomitantly, experimental and observational evidences endorse a significant role for the environment, maybe in the primary selection but, in any case, in the maintenance of the phenotype in the field, as the trade-offs associated with P. columella resistance are compensated by their higher tolerance to the more restrained environmental conditions for other competitor snails that comes with lower pH/TH values. This could be particularly significant if a disruptive or even a directional selection of resistance took place, as the fitness cost associated with P. columella resistance is unlikely to be relatively weak given the limited distribution of these populations in nature in spite of their ancient presence in the island.

In any case, while resistant populations have been found so far in Cuba, it might be considered that the primary features of these populations could have been selected somewhere in North America from where P. columella is suggested to have originated. However, no exhaustive sampling towards tracking this resistant phenotype has ever been carried out in this region.

The figure 1 aimed at providing an integrative synthesis of the main phenotypic features of resistant and susceptible P. columella to F. hepatica and the main results and hypothesis derived from comparative molecular and mechanistic immunobiological, ecological and evolutionary approaches applied to this system.

Figure 1. Comprehensive view of the main phenotypic features of resistant and susceptible

Pseudosuccinea columella to Fasciola hepatica and the main results and hypothesis derived from comparative molecular and mechanistic immunobiological, ecological and evolutionary approaches applied to this system. Symbols in circles refers to enhanced (+), diminished (-), and cancelled (x) pathways.

Future experimental perspectives: where we go from here

There is a growing interest for understanding immunobiological interactions within vector snails as they can increase our comprehension of innate defences from mechanistic and evolutionary points of view, of the dynamic of parasite transmission and of the multiple factors influencing parasite circulation. In the case of P. columella -F. hepatica, future studies aiming at deciphering the particular immune pathways and molecules involved during resistant/susceptible host-parasite interactions are essential to shed light on the differential determinants mediating each phenotype and to propose global mechanisms of resistance/infection for this system. Multiple "omics" approaches coupled with functional validation (RNA interference: siRNA, dsRNA) of the identified pathways following parasite exposure need to be applied to decipher the molecular and metabolic scenario in susceptible and resistant snails. In this sense, it will be also interesting to established the inheritance pattern of the identified specific markers through mating experiments, for theoretical and practical uses. However, as self-crossing constitutes the almost exclusive reproductive strategy of P. columella [START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF][START_REF] Lounnas | Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at the worldwide scale[END_REF], it is likely that directed castration should be put in place to resolve this issue.

Due to the association of the resistant phenotype with an increase molecular and phenotypical competence to cope with stress (Alba et al., 2019a[START_REF] Alba | Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba[END_REF], experimental infections on stressed-snails could be worth testing in this model. Environmental factors such as an increase of temperature and starvation are known to affect snail -parasite interaction by decreasing immune capabilities of the host [START_REF] Leicht | An experimental heat wave changes immune defense and life history traits in a freshwater snail[END_REF][START_REF] Seppälä | Maintenance of genetic variation in Immune defense of a freshwater snail: role of environmental heterogeneity[END_REF], increasing their susceptibility to digenean infections [START_REF] Leicht | Infection success of Echinoparyphium aconiatum (Trematoda) in its snail host under high temperature: role of host resistance[END_REF] and, even reverting certain resistant phenotypes (Ittiprasert andKnight, 2012, Knight et al., 2016). In this resistant P. columella model, the exposure to heat waves, low pH/TH and low food income prior infection could negatively affect the energetic budget necessary for the sustainability of its enhanced immune competence following F. hepatica exposure. Meanwhile, it would be worth exploring if housing susceptible snails in low pH/TH conditions could decrease their sensitivity to F. hepatica as it could strengthen the argument towards an environmentalmediated resistance to the parasite. Furthermore, experimental studies assessing life traits and competing capacities of resistant populations reared in sympatry with other freshwater snail species at common pH/TH conditions could gauge the cost of resistance against the most frequently found gastropods in Cuba.

An interesting interaction to approach in the future in this model would be the possibility of P. columella of actively avoiding F. hepatica infection. Recent studies depicting a scenario referred to as 'a landscape of disgust' explain how parasite avoidance shapes the ecology and evolution of host-parasite interactions [START_REF] Weinstein | A landscape of disgust[END_REF]. Heavily miracidia-infested areas in portions of ponds, rivers or other freshwater bodies may prevent P. columella of settling, and remain in distal regions with slightly different physical conditions. This, however, would be an interesting fact to explore given that snails usually become infected passively and have little to none chance of avoidance by a direct change in behavior.

Furthermore, field and experimental measures to characterize the encounter filter of resistant P. columella (e.g. encounter rates with F. hepatica, attractiveness of F. hepatica miracidia) could be also a motivating research with implications not only for biological sciences but also for epidemiology.

In addition, experimental evolution over few generations of parasite resistance on susceptible P. columella by selectively pressing with F. hepatica or with low pH/TH housing conditions might shed lights on the mechanistic bases behind the phenotypic features recorded in resistant populations. It could be also interesting to evaluate the sensibility of resistant and susceptible P. columella populations to other trematode parasites including those phylogenetically close F. hepatica and compatible with P. columella (e.g. Fasciola gigantica and Fascioloides magna) to deeply characterize the specificity of the resistance. All of these approaches deserve further investigation.

Acknowledgments

Authors would like to thank the editors Dr. Joanna Bridger and Dr. Mathilde Knight for the kind invitation to contribute to the special issue.

Funding statement

Partial financial support for this investigation was provided by the subventions granted to AA by the French Embassy in Cuba and to AAV by Institut de Recherche pour le Développement (BEST grant).

This study is set within the framework of the "Laboratoires d'Excellences (LABEX)" TULIP (ANR-10-LABX-41).

BG was supported by ANR JCJC INVIMORY (number ANR 13-JSV7-0009) from the French National Research Agency (ANR).

Authors contribution statement

Investigation

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mario Piedrahita-Bello

Yue Zan

Alejandro Enriquez-Cabrera

Gábor Molnár

Bertrand Tondu

Lionel Salmon
email: lionel.salmon@lcc-toulouse.fr

Azzedine Bousseksou
email: azzedine.bousseksou@lcc-toulouse.fr

Effect of the spin crossover filler concentration on the performance of composite bilayer actuators

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

Introduction

In the past few years, there have been an increasing interest in the use of ferrous spin crossover (SCO) complexes for mechanical transduction [1][2][3], exploiting the large, reversible volume expansion (up to 10 -15 %) [4][5][6][7] that accompanies the spin transition phenomenon when going from the low spin (LS) to the high spin (HS) state of the central metal ion [START_REF]Spin Crossover in Transition Metal Compounds I[END_REF][START_REF] Halcrow | Spin-Crossover Materials: Properties and Applications[END_REF]. This property allows SCO materials to transduce into mechanical work the various stimuli (thermal, optical, chemical …) that can trigger the SCO phenomenon. Previous work in this line of research has seen significant progress, going from an initial proof of concept [3] to electrothermally driven actuators [START_REF] Gural'skiy | [END_REF], sensors [11][12][13], generators [14][15], microactuators [16][17][18][19][20] and 3D-printed macroscopic actuators [21].

In this context, we have recently synthesized high quality, homogeneous SCO@P(VDF-TrFE) composites (P(VDF-TrFE) = poly(vinylidene fluoride-trifluoro-ethylene) using a blade casting technique. When combined with a second layer of Ag@P(VDF-TrFE) a bending bilayer actuator was obtained, which can be actuated by Joule effect [22]. By implementing a closed-loop control, these devices turned out to be very efficient, in terms of work output (>4 J/cm 3 ), controllability (>99% accuracy), life cycle (> 36000) and versatility, via different particle compositions and morphologies. These results provide a tangible scope for the use of SCO particle based polymer composites for applications in soft robotics and related fields.

In the present work, we discuss novel results towards the optimization of the work output of these bilayer actuators. Notably, we have investigated the relationship between the concentration of the SCO filler in a composite actuator and the actuation properties, including deflection amplitude, speed and controllability. To this aim we fabricated a series of bilayer actuators based on the [Fe(NH2trz)3]SO4 SCO complex (NH2trz = 1,2,4-4-NH2-triazole) [21,[23][24], modifying the proportion of SCO material in the active layer with respect to the initially used 25% SCO filler [22]. Compounds from this family forming 1-D polymeric chains are often studied because they present versatile properties in terms of transition temperature with the possibility to be nanostructured while maintaining interesting spin crossover properties [25][26][27].

The study of the filler concentration is obviously a key parameter because it allows to modulate not only the actuating properties, but also, in certain cases, the thermomechanical and/or the electrical properties of the active layer. For example, it was shown that the increase of quartz concentration in a dielectric elastomer can enhance the dielectric properties, resulting in an increased actuation amplitude [28]. Similar improvement was obtained while increasing the magnetic filler concentration in magnetic polymer composites, resulting also in the increase of the material stiffness [START_REF] Jayaneththi | IEEE Int. Conf. Adv. Intell. Mechatronics[END_REF]. For certain composite materials, however, the improvement of the actuation properties by the increase of the active filler can be detrimental to the mechanical (strength, stiffness,..) and/or electrical (percolation limit, charge accumulation,..) properties of the device and an optimal filler concentration have to be established to achieve a compromise. This effect can be illustrated for instance by the spontaneous decrease of the magnitude of the actuation stroke beyond 2 wt % loading of carbon nanotubes in polymer-nanotube composites [START_REF] Ahir | [END_REF].

Materials and methods

All solvents and reagents were purchased from Sigma Aldrich and used without further purification. The synthesis and characterization of the spin crossover complex [Fe(NH2trz)3]SO4 has been previously reported [21]. For the fabrication and characterization of the bilayer actuator devices, we followed the same methodology as described in ref. [22].

The SCO@P(VDF-TrFE) composite films were prepared by dispersing the SCO complex [Fe(NH2trz)3]SO4 (45, 90, 120 and 180 mg for 15, 25, 33 and 50 wt % composites, respectively) in 2-butanone (1.8 mL) in an ultrasonic bath for 40 min. Then, the P(VDF-TrFE) 70/30 copolymer (270, 270, 240 and 180 mg for 15, 25, 33 and 50 wt % composites, respectively) was added to the mixture and dissolved at 45 °C. The resulting suspensions were then bladecast at a height of 1.5 mm on a heated Teflon surface at 50 °C and kept at this temperature for 10 minutes, until the composite was dry. On top of this first layer, an electrically conductive layer was then prepared. For this, we dispersed 10 µm Ag flakes (318 mg) in 2-butanone (1.8 mL) in an ultrasonic bath for 5 min. Then, the P(VDF-TrFE) copolymer (216 mg) was added to the mixture and dissolved at 45 °C. The resulting suspensions were then blade-cast at a height of 1.5 mm on top of the dry SCO@P(VDF-TrFE) layer at 50 °C and kept at this temperature for ca. 2 h, until the composite was completely dry. The bilayer films were then annealed at 105 °C for 12 h. Bilayer devices with size 30 x 20 x 0.15 mm 3 were then cut into a U-shape (Fig. 1a) using a Realmeca RV 2-SP high precision lathe by attaching the bilayer films to an aluminum support. Scanning electron microscopy (SEM) images were acquired using a JEOL JSM 7800F Prime instrument operated at 5 kV. Samples for SEM were prepared by breaking the film cooled by liquid nitrogen and the cross-section was metalized with Pt. Variable temperature (0.5 °C/min) optical reflectivity measurements on all bilayers were conducted simultaneously with the analysis of the bulk SCO powder using a stereomicroscope (SMZ-168, Motic) equipped with a color camera (MOTICam 1000) and a heating-cooling stage (Linkam THMS-600). For the actuator control experiments, a Keithley 2420 source-meter unit was used to provide the input current for electro-thermal actuation. Position tracking of the tip bending of the actuator devices was performed using a Micro-Epsilon opto-NCDT 2300 laser triangulation system (Fig. 1b). The LabVIEW software was used both for open-and closed-loop control. The latter was achieved by means of conventional PID control [22]. The temperature of the samples was mapped via a Micro Epsilon 640 Thermal Imager.

Results and discussion

In agreement with previous reports [21], the obtained [Fe(NH2trz)3]SO4 complex presents a thermal spin transition at 73 °C and 61 °C on heating and cooling, respectively, as shown by the thermal variation of its optical reflectivity (Fig. 2a). We charged the P(VDF-TrFE) matrix by the microcrystalline powder of [Fe(NH2trz)3]SO4 at four different concentrations of the SCO material: 15 (1), 25 (2), 33 (3) and 50 (4) wt%. As shown in Figure 2a, for each composite sample, the thermal variation of the optical reflectivity indicates similar spin crossover properties, albeit at somewhat lower temperatures (ca. 70 and 56 °C) when compared to the bulk powder. Such differences are not unusual in SCO@polymer composites [31] and are usually ascribed to particle-matrix interactions and/or to different heat transfer in the composite (vs. the powder). On the other hand, the particle concentration has no substantial impact on the SCO curves in the investigated range. SEM images acquired on the bilayer crosssections are shown in Figures 2b-2e and Figure S1 in the Electronic Supporting Information (ESI). The separation of the two layers is neat, yet the common polymer backbone allows to maintain the mechanical integrity of the bilayer over numerous actuation cycles. Indeed, we have never encountered delamination, which is usually the major flaw of this type of bending bilayer devices. One can clearly distinguish also the rod-shaped SCO particles (ca. 1 micron mean length), which appear homogeneously distributed in the matrix for each particle concentration without notable preferential orientation (see Fig. 2 andESI). The open-loop free actuating behavior of the four actuators is shown in Figure 3a, which displays the cantilever tip displacement as a function of time in response to the highest current bias, which can be applied to the sample without damaging it, 1.3 A for sample 1, 0.95 A for sample 2, 1.25 A for sample 3 and 1.1 A for sample 4 (see Fig. S2 for the detailed open loop measurements). The maximum actuation amplitude of 1 is 4 mm and of 2 is 5.5 mm. It is important to notice that we have reproduced the preparation of sample 2 (sample 2'), which presents very similar behavior with a maximum actuation amplitude of 5 mm (see Fig. S2), albeit for somewhat different applied bias (1.05 A) due to the slightly different resistivity of the silver layer. As it might be expected, the actuation range of 3 is significantly higher (8 mm), which represent a ca. 50 and 100 % increase with respect to 2 and 1. This increment is roughly proportional to the increase of the SCO filler weight fraction. On the other hand, the actuation range of 4 is somewhat lower than that of 3 despite the increase of the SCO concentration to 50 %. This finding denotes that there is a threshold concentration after which adding more active material does not lead to the increase of the actuation range. Most likely, for such high concentrations the homogeneity of the composite becomes compromised and particleparticle interactions tend to gain increasing role in the mechanical behavior of the composite (see SEM images in ESI). In open loop conditions, the steady-state position is reached in ca. 30-60 s for each actuator. Interestingly, one can notice a downward motion for actuators 3 and 4 at the start of the movement, which contributes to the increase of the response time.

In the case of actuator 4, this reverse motion is very important, with the actuator moving ca.

0.8 mm in the opposite direction before starting the actuation in the proper direction (see Fig. 3a). This downward motion seems to be caused by the intrinsic modification of the mechanical properties of the composite amplified by the increasing SCO concentration. At this point, it is worth to underline that a quantitative link between the SCO behaviour (Fig. 2a) and the actuating response (Fig. 3) is difficult to establish. Indeed, the spin transition in such Jouleheated bending actuators is intrinsically heterogeneous due to the inhomogeneous temperature distribution -as illustrated by the thermal mapping of the SCO layer of the actuator 2' upon increasing current bias (Fig. S3), obtained with an IR camera. To further analyse the concentration effect, actuators 2, 3 and 4 were actuated in closed-loop conditions (without payload) to perform a fixed 5 mm displacement (Fig. 3b). The PID parameters were hand-tuned to reach an optimum value. Note that the maximum displacement of actuator 1 is only 4 mm, so the closed-loop behaviour of the latter is shown in a separate figure (Fig. S4). As it has been already shown in our previous work [22], a remarkable, ca. 1 order of magnitude improvement of the response time is obtained upon closed-loop control (vs. the open-loop values). (We define the response time as the time it takes for the actuator to reach the 5% error range of a given target position and remain stable inside this range). Notably, the closed-loop response time of actuators 1-3 falls in the range between 1.5 -3 s. On the other hand, a significant slowdown of the closed-loop response is observed for actuator 4, which is associated with a highly oscillatory response, compromising thus the controllability of the system.

Closed loop actuation of the samples while bearing a payload of 204 mg (approx. 3 times the actuator mass) is shown in Figures 4a-4b. The fixed displacement experiments with payloads (Fig. 4a and Fig. S4) reveal a significant "load robustness" in that the response of the actuators with payloads is comparable to their free actuation behavior discussed above. However, actuator 4, having a higher energetic inertia due to the higher proportion of the SCO material, shows a highly oscillatory response, denoting a significant loss of controllability. However, while comparing the reverse motion with and without payload for the different actuators, a significant increase (from 0.8 to 2 mm) is only observed for actuator 4 and seems to result from the concomitant effect of the reduce stiffness of the composite and its own weight. It is interesting to notice also that, unlike for the free actuation, the response time of actuator 3 (4.25 s) is somewhat higher than that of actuator 2 (3.45 s), but such small differences lie within the experimental uncertainty. In order to further test the controllability of these devices, the actuators were then operated with the payloads using a sinusoidal position input under closed-loop conditions. Figure 4c

Conclusions

In summary, we fabricated and characterized electro-thermally actuated, bending bilayer [Fe(NH 2 trz) 3 ]SO 4 @P(VDF-TrFE)/Ag@P(VDF-TrFE) actuators varying spin crossover filler concentrations. We have shown that the achievable actuating amplitude increases from ca. 4 to 8 mm when increasing the SCO filler fraction from 15 to 33 %, while keeping similar response times (ca. 30-60 s in open-loop and 2-3 s in closed-loop) and similar load-bearing capacity. On the other hand, a further increase of the SCO content to 50 % does not afford for higher amplitude movements and leads to reduced response times, which we attributed to the combined effects of the increased energetic barrier (for actuation) and reduced sample homogeneity. We expect that other SCO@polymer composite actuators will show a similar tendency for an optimum concentration, whose value must be determined case-by-case.

Indeed, the actuation performance is determined by a complex interplay between several parameters, such as the particle morphology, the device geometry, elastic and thermal properties of the materials and so forth, which must be optimized conjointly.

Fig. 1 .

 1 Fig. 1. Bilayer actuator devices. a) Photograph of an actuator device showing the grey conductive layer (left) and the pink SCO layer (right). b) Scheme of the actuator test bench indicating the Joule heating, the optical displacement detection and the PID control. c) Actuators composition.

Fig. 2 .

 2 Fig. 2. SCO composites. a) Variable temperature optical reflectivity acquired simultaneously for the bulk powder of [Fe(NH2trz)3]SO4 and for the [Fe(NH2trz)3]SO4@P(VDF-TrFE) composites with different particle concentrations.(Note that the abrupt change of reflectivity refers to the SCO phenomeon, whereas the more gradual reflectivity changes at higher temperatures correspond to the Curie transition in the copolymer matrix -with increasing proportion for decreasing SCO load.). Selected SEM images of the SCO@polymer cross-sections for different particle concentrations b) 15%, c) 25%, d) 33% and e) 50%.

Fig. 3 .

 3 Fig. 3. Open-and closed-loop actuation behavior. a) Highest amplitude open-loop and b) fixed (5 mm) amplitude closed-loop actuation of the [Fe(NH2trz)3]SO4 bilayer actuators fabricated

and 4d shows these experiments for actuators 2 and 4 ,

 4 respectively. In each case, the movement closely mirrors the given input, with a positioning error, which remains generally below 5 %. It is clear that the two systems retain high controllability once the initial energetic barrier has been cleared, showing that the difference in controllability and performance between the actuators fabricated at different concentrations mostly pertains their actuation range and their behavior during the initial period of the actuation.

Fig. 4 .

 4 Fig. 4. Closed-loop actuation with payloads. a) Closed-loop step-wise actuation (target position = 5 mm) of the [Fe(NH2trz)3]SO4 bilayer actuators with payload for different SCO filler concentrations (25, 33 and 50 %). Horizontal dotted lines indicate the 5% error range and vertical dotted lines indicate the response time of the actuators. b) Photographs of a bilayer device (25 wt%) with a payload of 204 mg showing the bending movement between the LS and HS states. (c-d) Closed-loop sinusoidal actuation of the actuators with payload for (c) 25 wt% and (d) 50 wt% of SCO filler concentration.

Funding This work received financial support from the Federal University of Toulouse/ Région Occitanie (PhD grant of MPB) and from the Agence Nationale de la Recherche (ANR-19-CE09-0008-01).