Mariana G Lima

Ronaldo De C Augusto

Jairo Pinheiro

Silvana C Thiengo

Physiology and immunity of the invasive giant African snail, Achatina (Lissachatina) fulica, intermediate host of Angiostrongylus cantonensis

Keywords: Achatina fulica, Immunology, Physiology, Nematodes, Angiostrongylus cantonensis

As one of the most successful invasive land snail species, Achatina (Lissachatina) fulica Bowdich, 1822 has achieved wide global distribution, particularly in (sub)tropical regions, with further dispersal likely due to climate change. This species of giant African snails (up to 17 cm shell length) is a pest that has extensive negative impact on agriculture and can serve as vector for several parasites, including Angiostrongylus cantonensis, a nematode parasite that causes (human) eosinophilic meningitis, an emergent disease. Investigation showed that A. cantonensis infection negatively impacts the metabolism of A. fulica by depleting polysaccharide stores of the intermediate host, compromising the energy balance of the snail. A review of the literature indicates that A. fulica possesses potent innate type immune defenses to counter infection, including phagocytic hemocytes capable of deploying reactive oxygen species and lectins for non-self recognition, a serine protease-dependent coagulation response (not observed in other taxa of gastropods), as well as antimicrobial proteins including achacin, an antimicrobial protein. A recent chromosome level genome assembly will facilitate progressively detailed characterization of these immune features of A. fulica. We strongly encourage further immunological studies of A. fulica, ranging from organismal level to molecular biology to gain better understanding of the A. fulica internal defense response to nematode pathogens like A. cantonensis and the contribution of immune function to the invasiveness of (snail) species. Characterization of immunity of A. fulica, representing the understudied Stylommatophora (panpulmonate landsnails) will also broaden the comparative immunology of Gastropoda.

Introduction

Gastropoda comprises the largest Class of Mollusca, occurring in a wide variety of aquatic and terrestrial habitats, showing a great diversity in anatomical structure. Accordingly, gastropods have contended with many diverse pathogens, including opportunists and several phylogenetic lineages of specialized eukaryotic parasites such as the digenetic trematodes [START_REF] Loker | Gastropod immunobiology[END_REF][START_REF] Aktipis | Gastropoda an overview and analysis[END_REF]. Considering the framework of great species richness and diversity of lifestyles, gastropod immunology is likely highly diverse due to lineagespecific evolution of novel features or modification and/or loss of ancestral aspects of immune function (e.g. [START_REF] Gorbushin | Derivatives of the lectin complement pathway in Lophotrochozoa[END_REF]. To date, however, immune function has been studied consistently in only a modest number of gastropod species (e.g. [START_REF] Schultz | Comparative immunogenomics of molluscs[END_REF] that does not validly represent the phylogenetic diversity of gastropods.

Achatina fulica, a prominent invasive pest

Importantly, comprehensive understanding of immunology is especially lacking for air breathing terrestrial slugs and snails that belong to the order Stylommatophora within the clade of Eupulmonata (Gastropoda, Euthyneura, Heterobranchia). The stylommatophorans comprise of Scolodontidae (a small basal group) and two large clades of non-achatinoid and achatinoid snails (e.g. [START_REF] Wade | Evolutionary relationships among the Pulmonate land snails and slugs (Pulmonata, Stylommatophora)[END_REF][START_REF] Saadi | Resolving the basal divisions in the stylommatophoran land snails and slugs with special emphasis on the position of the Scolodontidae[END_REF]. The latter clade includes the family Achatinidae of medium to large sized tropical land snails, native to the African continent, that contains the subject of this review. Achatina (Lissachatina) fulica Bowdich, 1822. Currently A. fulica is included within the subgenus Lissachatina and some but not all recent publications (e.g. [START_REF] Borrero | Into the Andes: three new introductions of Lissachatina fulica (gastropoda, Achatinidae) and its potential distribution in South America[END_REF]GuoY et al., 2019) have treated Lissachatina as a genus; however, at present, no published taxonomic works are available to sustain this treatment. This review will employ the binomial Achatina fulica. This species, commonly named the giant east African snail grows to large adult size (shell length up to 17 cm). Like other giant African snails, A. fulica is considered a food source and a pet animal, and it may be applied in traditional medicine for antibacterial activity of secreted snail mucus [START_REF] Etm | Antibacterial properties of snail mucus on bacteria isolated from patients with wound infection[END_REF]. A few (or even single) A. fulica are capable of colonize new habitats; they are hermaphrodites that can selffertilize and have a high reproductive output. It is known to consume more than 50 species of tropical plants, agricultural and horticultural crops, modify habitats and outcompete native snails in newly colonized non-native ecosystems [START_REF] Raut | Achatina fulica Bowdich and other Achatinidae as pests in tropical agriculture[END_REF], by predation and herbivory. It is of note that A. fulica impacts humans severely as a general nuisance, since snails populations may reach very high densities [START_REF] Mead | Helicid land mollusks introduced into North America[END_REF][START_REF] Thiengo | Rapid spread of an invasive snail in South America: the giant African snail, Achatina fulica, in Brasil[END_REF][START_REF] Hoxha | Comparative feeding behaviour of native and introduced terrestrial snails tracks their ecological impacts[END_REF].

These factors, along with trends of cross-border migration and trading, have likely contributed greatly to transport and introduction of A. fulica outside traditional native distribution ranges, allowing this snail to emerge as an invasive alien species [START_REF] Knobler | The Impact of Globalization on Infectious Disease Emergence and Control: Exploring the Consequences and Opportunities: Workshop Summary[END_REF]. While fortunately, it seems that only a modest proportion of introduced organisms possesses the phenotypic plasticity of relevant traits for behavior and fitness needed to become an invasive species [START_REF] Richardson | and invasion of alien plants: concepts and definitions[END_REF][START_REF] Richardson | Plant invasions: merging the concepts of species invasiveness and community invasibility[END_REF], several species of terrestrial gastropods have been able to invade and establish in a wide variety of new ranges, including natural zones like the Amazon Rainforest and large urban agglomerations worldwide [START_REF] Ruiz | In: Invasive Species: Vectors and Management Strategies[END_REF][START_REF] Roda | Reproductive ecology of the giant African snail in South Florida: implications for eradication programs[END_REF][START_REF] Hoxha | Comparative feeding behaviour of native and introduced terrestrial snails tracks their ecological impacts[END_REF]. Classified among the 100 worst invasive pests in the world [START_REF] Lowe | The Invasive Species Specialist Group (ISSG) a Specialist Group of the Species Survival Commission (SSC) of the World Conservation Union (IUCN)[END_REF], A. fulica is considered the most widely introduced and invasive land snail species pest with particular impact in tropical and subtropical regions. This may be exacerbated by climate change as this may lead to range expansion of invasive species and interact synergistically with invaders to further worsen the impact on local ecosystems [START_REF] Rekha | Effect of climate change on invasion risk of giant African snail (Achatina fulica Férussac, 1821: Achatinidae) in India[END_REF];[START_REF] Bellard | Impacts of climate change on the future of biodiversity[END_REF][START_REF] Lowe | The Invasive Species Specialist Group (ISSG) a Specialist Group of the Species Survival Commission (SSC) of the World Conservation Union (IUCN)[END_REF][START_REF] Raut | Achatina fulica Bowdich and other Achatinidae as pests in tropical agriculture[END_REF]. Concerning the invasive colonization of the Americas, A. fulica was initially introduced from Hawaii (where it persists to date) to Florida, mainland of the USA in 1966 [START_REF] Robinson | Alien invasions: the effects of the global economy on non-marine gastropod introductions into the United States[END_REF][START_REF] Kim | Diverse gastropod hosts of Angiostrongylus cantonensis, the rat lungworm, globally and with a focus on the Hawaiian Islands[END_REF]. In the Caribbean, A. fulica was established by 1984 on the island of Guadeloupe, by 1988 on Martinique and subsequently on Barbados and Saint Lucia according to [START_REF] Raut | Achatina fulica Bowdich and other Achatinidae as pests in tropical agriculture[END_REF]. More recently, A. fulica was reported from Jamaica and Haiti by [START_REF] Lindo | Enzootic Angiostrongylus cantonensis in rats and snails after an outbreak of human eosinophilicmeningitis[END_REF] and [START_REF] Raccurt | Présence d'Angiostrongylus cantonensis en Haïti[END_REF], respectively. Since initial reports from the 1980s, A. fulica has been reported from many countries in South America including Argentina [START_REF] Gutiérrez Gregoric | Invasion of the Argentinean paranense rainforest by the giant African snail Achatina fulica[END_REF], Colombia [START_REF] De | Registro del caracol africano gigante Achatina fulica (Bowdich 1822) (Mollusca: gastropoda-Achatinidae) en Sincelejo, costa Caribe de Colombia[END_REF]; Ecuador [START_REF] Correoso | Estrategia preliminar para evaluar y erradicar Achatina fulica (Gastropoda: Achatinaceae) em Ecuador[END_REF], Paraguay [START_REF] Vogler | The giant African snail, Achatina fulica (Gastropoda: Achatinidae): using bioclimatic models to identify South American áreas susceptible to invasion[END_REF], Peru [START_REF] Borrero | Into the Andes: three new introductions of Lissachatina fulica (gastropoda, Achatinidae) and its potential distribution in South America[END_REF] and Venezuela [START_REF] Martínez-Escarbassiere | Nota acerca de la Achatina (Lissachatina) fulica (Bowdich,1822) peligroso caracol africano (Pulmonata-Achatinidae) introduzido en Venezuela[END_REF]. In Brazil, A. fulica was first introduced deliberately in 1988 in the southern state of Paraná [START_REF] Teles | Implicacões da introdução e dispersão de Achatina fulica Bowdich, 1822 no Brasil[END_REF], where snails (probably from Indonesia) were sold at an agricultural fair as food resource for human consumption. Nowadays, A. fulica occurs in 25 out of the 26 states and the Federal District of Brazil [START_REF] Morassutti | Eosinophilic meningitis caused by Angiostrongylus cantonensis: an emergent disease in Brazil[END_REF]Thiengo andFernandez, 2013, 2016).

Achatina fulica is very difficult to control once established; eradication has proven virtually impossible, especially in developing countries. In excess of $1 million dollars were spent for (ongoing) counter measures against A. fulica during the initial five years after this invasive snail species established in Florida (USA), (see e.g. [START_REF] Mead | Helicid land mollusks introduced into North America[END_REF][START_REF] Prociv | Neuro-angiostrongyliasis: unresolved issues[END_REF][START_REF] Raut | Achatina fulica Bowdich and other Achatinidae as pests in tropical agriculture[END_REF][START_REF] Thiengo | The giant African snail Achatina fulica as natural intermediate host of Angiostrongylus cantonensis in Pernambuco, northeast Brazil[END_REF]. To date, it is illegal to import or keep A. fulica in the USA (see https://www.aphis. usda.gov/aphis/ourfocus/planthealth/plant-pest-and-diseaseprograms/pests-anddiseases/giant-african-snail/gas) and control efforts include chemical phytosanitary treatment of agricultural products to be shipped out of quarantined areas. Molluscicide treatments of A. fulica habitats can significantly inhibit snail development and reduce transmission of the parasites that these snails harbor (Gomot-de [START_REF] Vaufleury | Methods for toxicity Assessment of contaminated soil by oral or Dermal uptake in land snails. 1. Sublethal effects on growth[END_REF]. Unfortunately, the efficacy of molluscicides varies [START_REF] Hallman | Phytosanitary Applications of irradiation[END_REF][START_REF] Hallman | Phytosanitary irradiation of the invasive herbivorous terrestrial snail Cornu aspersum (Stylommatophora: Helicidae)[END_REF], and intensive use has a negative environmental impact as it leads to chronic soil contamination [START_REF] Jansch | Effects of Pesticides on Soil Invertebrates in Model Ecosystem and Field Studies: a Review and Comparison with Laboratory Toxicity Data[END_REF][START_REF] Arias-Estévez | The mobility and degradation of pesticides in soils and the pollution of groundwater resources[END_REF][START_REF] Thiengo | Rapid spread of an invasive snail in South America: the giant African snail, Achatina fulica, in Brasil[END_REF].

Achatina fulica, vector of parasitic nematodes

Another negative aspect of invasion of new ranges by A. fulica is that this snail may transmit or even introduce pathogens with undesirable health and socioeconomic effects. Like several other species of land snails that harbor parasites, e.g the widely distributed brown garden snail Cornu aspersum (Müller, 1774; formerly Helix aspersa), vector of the nematodes Angiostrongylus vasorum and Aelurostrongylus abstrusus [START_REF] Druart | Snails as indicators of pesticide drift, deposit, transfer and effects in the vineyard[END_REF][START_REF] Di Cesare | Larval development of Angiostrongylus vasorum in the land snail Helix aspersa[END_REF]; Theba pisana (Müller 1774), the white garden snail present in Australia, Israel, and USA), intermediate host for the nematode lungworm Muellerius capillaris and the trematode Brachylaima cribbi [START_REF] Baker | The Biology and Control of White Snails (Mollusca, Helicidae), Introduced Pests in: Australia. Commonwealth Scientific and Industrial Organization[END_REF][START_REF] Butcher | Description of the life-cycle stages of Brachylaima cribbi n. sp. (Digenea: Brachylaimidae) derived from eggs recovered from human faeces in Australia[END_REF][START_REF] Grewal | Parasitism of molluscs by nematodes: types of associations and evolutionary trends[END_REF], also A. fulica snails may spread plant pathogens and they are intermediate host of several parasitic nematodes, such as Aelurostrongylus abstrusus, Phasmarhabditis hermaphrodita, Rhabditis sp, Strongyluris sp [START_REF] Turner | Snail transmission of species of Phytophthora with special reference to foot rot of piper nigrum[END_REF][START_REF] Alicata | The discovery of Angiostrongylus cantonensis as a cause of human eosinophilic meningitis[END_REF][START_REF] Zanol | O caramujo exótico invasor Achatina fulica (Stylommatophora, Mollusca) no Estado do Rio de Janeiro (Brasil): situação atual[END_REF][START_REF] Williams | Susceptibility of the Giant African snail (Achatina fulica) exposed to the gastropod parasitic nematode Phasmarhabditis hermaphrodita[END_REF][START_REF] Oliveira | Distribution of cysts of Strongyluris sp. (nematoda) in the palial system of Achatina fulica Bowdich, 1822 from Vila Dois Rios and Vila do Abraão, Ilha Grande, Angra dos Reis, Rio de Janeiro[END_REF]. Importantly, A. fulica is also vector of two zoonotic nematodes: Angiostrongylus costaricensis, causing abdominal angiostrongyliasis and the congeneric rat lungworm, Angiostrongylus cantonensis, which causes eosinophilic meningoencephalitis (or the rat lungworm disease) in humans [START_REF] Alicata | The discovery of Angiostrongylus cantonensis as a cause of human eosinophilic meningitis[END_REF][START_REF] Prociv | Neuro-angiostrongyliasis: unresolved issues[END_REF][START_REF] Carvalho | Potentiality of Achatina fulica Bowdich, 1822 (Mollusca: gastropoda) as intermediate host of Angiostrongylus costaricensis Morera and Céspedes[END_REF][START_REF] Carvalho | Angiostrongylus cantonensis (Nematode: Metastrongyloidea) in molluscs from harbour areas in Brazil[END_REF]. Abdominal angiostrongyliasis appears to be restricted to the Americas, with reports ranging from the southern United States to northern Argentina [START_REF] Graeff-Teixeira | Expansion of Achatina fulica in Brazil and potential increase risk for angiostrongyliasis[END_REF]. Originally endemic in Southeast Asia and Pacific Islands, to date, eosinophilic meningitis has been reported from about 30 countries of all continents with exception of Europe and Antarctica [START_REF] Cowie | Eosinophilic meningitis caused by Angiostrongylus cantonensis, the rat lungworm: biology, distribution, epidemiology, detection, diagnosis, treatment, and management[END_REF]. In Hawaii, A. fulica has been associated with transmission of eosinophilic meningoencephalitis [START_REF] Kim | Diverse gastropod hosts of Angiostrongylus cantonensis, the rat lungworm, globally and with a focus on the Hawaiian Islands[END_REF].

The life cycle of A. cantonensis involves rodents as definitive hosts and several species of terrestrial and freshwater snails that may serve as intermediate hosts. Gastropods become infected by ingestion of rodent feces that contain A. cantonensis larvae (L1 stage). Inside the snail, the parasite molts twice over about 15 days. The resulting L3 larvae are directly infective for the definitive host [START_REF] Cheng | On the mode of infection of Achatina fulica by the larvae of Angiostrongylus cantonensis[END_REF][START_REF] Maldonado | First report of Angiostrongylus cantonensis (nematoda: Metrastrongylidae) in Achatina fulica (Mollusca: gastropoda) from Southeast and South Brazil[END_REF] but an optional role for paratenic (carrier) hosts such as land crabs, freshwater prawns, fish, frogs and planarians increases the chances for transmission of the parasite. Angiostrongylus costaricensis has a similar life cycle but does not employ paratenic hosts [START_REF] Morera | Life history and redescription of Angiostrongylus costaricensis Morera and Céspedes, 1971[END_REF][START_REF] Spratt | Species of Angiostrongylus (nematoda: Metastrongyloidea) in wildlife: a review[END_REF].

Human Angiostongylus infections result from ingestion of raw gastropods or paratenic hosts (in case of A. cantonensis) that harbor L3 nematode parasites, or by food that was contaminated with L3 larvae from decayed, disrupted snail bodies or perhaps shed from infected snails [START_REF] Graeff-Teixeira | Update on eosinophilic eningoencephalitis and its clinical relevance[END_REF]. Because humans are not natural definitive hosts, neither Angiostrongylus species is able to complete their life cycle; parasite eggs or larvae are not released into the feces and this makes diagnosis difficult. Although the parasites do not survive long in humans, infection causes uncomfortable symptoms including nausea, vomiting, neck stiffness, fever and headaches. Occasionally, patients develop complications that lead to intestinal obstruction and perforation (A. costaricensis) or neurological damage (A. cantonensis) and even death.

Both A. costaricensis and A. cantonensis show low specificity for intermediate hosts; many different species of terrestrial and aquatic (fresh water) gastropods with natural Angiostrongylus infection have been observed around the world (Wallace and Rosen, 1969a;[START_REF] Malek | Medical and Economic Malacology[END_REF][START_REF] Carvalho | Angiostrongylus cantonensis (Nematode: Metastrongyloidea) in molluscs from harbour areas in Brazil[END_REF][START_REF] Thiengo | The giant African snail Achatina fulica as natural intermediate host of Angiostrongylus cantonensis in Pernambuco, northeast Brazil[END_REF][START_REF] Kim | Diverse gastropod hosts of Angiostrongylus cantonensis, the rat lungworm, globally and with a focus on the Hawaiian Islands[END_REF]. The introduced snail species A. fulica and South American ampullariid Pomacea canaliculata (Lamarck, 1822) are the main intermediate hosts of A. cantonensis in the south of China [START_REF] Lv | The emergence of angiostrongyliasis in the People's Republic of China: the interplay between invasive snails, climate change and transmission dynamics[END_REF], and an extensive list of molluscs transmits angiostrongyliasis on the Hawaian Islands [START_REF] Kim | Diverse gastropod hosts of Angiostrongylus cantonensis, the rat lungworm, globally and with a focus on the Hawaiian Islands[END_REF]. From Brazil, reports of natural intermediate host molluscs [START_REF] Morassutti | Eosinophilic meningitis caused by Angiostrongylus cantonensis: an emergent disease in Brazil[END_REF][START_REF] Thiengo | Moluscos como Hospedeiros intermediários de Angiostrongylus cantonensis en Brasil[END_REF] include various terrestrial pulmonates (Stylommatophora): Belocaulus willibaldoi Ohlweiler, Mota andGomes, 2009, Bradybaena similaris (Férussac, 1821), Sarasinula linguaeformis (Semper, 1885) (=Sarasinula marginata according to Thomé, 1989), Subulina octona (Bruguière, 1792) and A. fulica. Transmission of A. cantonensis was also reported for another ampullariid Pomacea lineata (Spix in Wagner, 1827), in northeast Brazil [START_REF] Thiengo | The giant African snail Achatina fulica as natural intermediate host of Angiostrongylus cantonensis in Pernambuco, northeast Brazil[END_REF].

In the laboratory, the life cycle of both A. cantonensis and A. costaricensis has been maintained through experimental infection of Biomphalaria glabrata (Say, 1818) a freshwater pulmonate snail [START_REF] Richards | Studies on Angiostrongylus cantonensis in molluscan intermediate hosts[END_REF][START_REF] Tunholi | Biochemical profile of Biomphalaria glabrata (Mollusca: gastropoda) after infection by Echinostoma paraensei (Trematoda: Echinostomatidae)[END_REF][START_REF] Bonfim | Biological and histological changes in reproductive patterns of Biomphalaria glabrata experimentally co-infected by Echinostoma paraensei and Angiostrongylus cantonensis[END_REF]. Taking in account the epidemiology of eosinophilic meningitis transmission, reports of A. fulica infected with A. cantonensis in 33 municipalities from 11 different Brazilian states [START_REF] Thiengo | Moluscos como Hospedeiros intermediários de Angiostrongylus cantonensis en Brasil[END_REF][START_REF] Cognato | First report of Angiostrongylus cantonensis in Porto Alegre, state of Rio Grande do Sul, southern Brazil[END_REF], and that the widely distributed A. fulica snails usually occur in dense populations which favors the contact of people with the parasite, A. fulica is considered the main vector for Angiostrongylus-associated eosinophilic meningitis in Brazil. Study of snail physiology, parasitology and immunity will likely provide inroads for improved or novel control strategies to extend the options to mediate the overall impact of invasive A. fulica.

Impact of nematode infection on Achatina fulica physiology

Study of the physiology of infected snails revealed the burden of A. cantonensis infection upon A. fulica. Regulation of energy metabolism is essential for biological functions and the maintenance of life [START_REF] Fraga | Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum[END_REF]. In pulmonate gastropods, including A. fulica, this regulation centers around availability of carbohydrates, deposited as glycogen and galactogen in special storage cells that are located in the mantle, among the digestive gland acini, the gonads, and also the muscular tissue of the cephalopedal mass [START_REF] Joosse | The hormone molluscs[END_REF][START_REF] Joosse | Trichobilharzia ocellata: physiological characterization of giant growth, glycogen deposit and absence of reproductive activity in the intermediate snail host, Lymnaea stagnalis[END_REF][START_REF] Tunholi-Alves | Activation of anaerobic metabolism in Biomphalaria glabrata (Mollusca:Gastropoda) experimentally infected by Angiostrongylus cantonensis (Nematoda, Metastrongylidae) by high-performance liquid chromatography[END_REF][START_REF] Tunholi | Aerobic to anaerobic transition in Biomphalaria glabrata (Say, 1818) infected with different miracidial doses of Echinostoma paraensei (Lie and Basch, 1967) by high-performance liquid chromatography[END_REF]Pinheiro and Amato, 1994a,b). During physiological stress conditions, such as caused by parasite infection, the carbohydrate reserves are mobilized to make glucose available as substrate to oxidative metabolism for production of ATP. This involves a complex activation process, including glycogenolysis and components of oxidative respiration; glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (TunholiAlves et al., 2014;[START_REF] Massa | High performance column liquid chromatographic analysis of selected carboxylic acids in Biomphalaria glabrata patently infected with Schistosoma mansoni[END_REF][START_REF] Bezerra | A comparative study of the organic acid content of the hemolymph of Schistosoma mansoni-resistant and susceptible strains of Biomphalaria glabrata[END_REF].

Alterations in this carbohydrate metabolism have been recorded from infected snails using different snail hostlarval parasite systems, and glycemia is generally reduced significantly [START_REF] Becker | Metabolic interrelationship of parasitic trematodes and molluscs, especially Schistosoma mansoni in Biomphalaria glabrata[END_REF]ElAnsary et al., 2000;[START_REF] Lima | The influence of Angiostrongylus cantonensis (Nematoda, Metastrongylidae) infection on the aerobic metabolism of Biomphalaria straminea and Biomphalaria tenagophila (Mollusca, Gastropoda)[END_REF][START_REF] Lima | Effects of experimental Angiostrongylus cantonensis infection on the reproductive biology of Biomphalaria straminea and Biomphalaria tenagophila[END_REF][START_REF] Martins | Bulimulus tenuissimus (mollusca) as a new potential host of Angiostrogylus cantonensis (nematoda), a histological and metabolic study[END_REF]. Larval helminths absorb glucose from snail hemolymph as substrate for ATP generation in support of the parasite metabolism and the development of intramolluscan larvae [START_REF] Becker | Metabolic interrelationship of parasitic trematodes and molluscs, especially Schistosoma mansoni in Biomphalaria glabrata[END_REF].

Demonstration of glycolytic enzyme activities provided evidence that oxidative pathways exist in larval A. cantonensis [START_REF] Shih | Glycolytic enzymes in juvenile and adult Angiostrongylus cantonensis[END_REF]. The competition by the parasite for nutrients causes a physiological state in the snail hosts that is similar to starvation [START_REF] Pinheiro | Physiological changes in Lymnaea columella (Say, 1818) (Mollusca, gastropoda) in response to Echinostoma paraensei Lie and Basch, 1967 (Trematoda, Echinostomatidae) infection[END_REF]. [START_REF] Brockelman | Carbohydrate reserves and hemolymph sugars of the African giant snail, Achatina fulica in relation to parasitic infection and starvation[END_REF] first described changes in the carbohydrate metabolism of A. fulica snails following experimental infection with A. cantonensis. Initially, the glucose content of the snail hemolymph was significantly reduced during the pre-patent infection, returning to normal values after one week. [START_REF] Tunholi-Alves | Unveiling the oxidative metabolism of Achatina fulica (Mollusca:Gastropoda) experimentally infected to Angiostrongylus cantonensis (Nematoda: Metastrongylidae)[END_REF] showed that reduction of glucose levels was associated with depletion of glycogen deposits in the digestive gland and cephalopedal mass of A. cantonensisinfected A. fulica. Along with similar observations from another parasitized snail species, B. glabrata harboring A. cantonensis [START_REF] Tunholi-Alves | Activation of anaerobic metabolism in Biomphalaria glabrata (Mollusca:Gastropoda) experimentally infected by Angiostrongylus cantonensis (Nematoda, Metastrongylidae) by high-performance liquid chromatography[END_REF], this demonstrates the existence of homeostatic mechanisms that rely on mobilization of tissue stores of carbohydrate (glycogen and galactogen) to recover and maintain glycemia levels in snail hemolymph. Parasite load (intensity of infection) influences the extent to which the oxidative metabolism of A. fulica is altered by A. cantonensis, as revealed by different degrees of impact on activities of hexokinase (HK) and lactate dehydrogenase (LDH), catalytic enzymes that are linked the oxidative metabolism of the snail host. The activity of HK, catalyst of the first step of glycolysis, increased significantly (relative to controls) when A. fulica was infected with ≥2500 (L1) A. cantonensis larva. Accompanied by reduced glucose levels, heightened HK activity reflects an increased energy (ATP) demand in the A. fulica host, warranted by the need to maintain basic vital processes in the presence of developing larval A. cantonensis [START_REF] Tunholi-Alves | Activation of anaerobic metabolism in Biomphalaria glabrata (Mollusca:Gastropoda) experimentally infected by Angiostrongylus cantonensis (Nematoda, Metastrongylidae) by high-performance liquid chromatography[END_REF]. Depending on parasite load, A. cantonensis causes yet additional subsequent changes in the oxidative metabolism of A. fulica.

Snails infected with fewer than 10,000 L1 larvae develop significant reductions of pyruvate and oxaloacetate. These changes may be due to either (I) accelerated redox reactions of the TCA cycle to support increased demand for ATP that challenge snail metabolism, causing a shortfall in the adequate supply of critical TCA intermediates, like oxaloacetate (substrate for the first reaction of the TCA cycle) or (II) the shunting of oxaloacetate away from the TCA cycle to drive gluconeogenesis for production of glucose using non-glycemic substrates in an attempt to restore normoglycemia [START_REF] Tunholi-Alves | Activation of anaerobic metabolism in Biomphalaria glabrata (Mollusca:Gastropoda) experimentally infected by Angiostrongylus cantonensis (Nematoda, Metastrongylidae) by high-performance liquid chromatography[END_REF]. Furthermore, A. fulica snails that received high infective doses (10,000 and 15,000 L1 larvae) of A. cantonensis [START_REF] Tunholi-Alves | Unveiling the oxidative metabolism of Achatina fulica (Mollusca:Gastropoda) experimentally infected to Angiostrongylus cantonensis (Nematoda: Metastrongylidae)[END_REF] showed significant increases in activity of LDH (key enzyme for anaerobic respiration), along with lactate accumulation and decreased levels of pyruvate in the hemolymph, indicating a transition of the oxidative metabolism from aerobic to anaerobic during infection, similar to results obtained with chromatographic and spectrophotometric methods for Biomphalaria harboring A. cantonensis [START_REF] Lima | The influence of Angiostrongylus cantonensis (Nematoda, Metastrongylidae) infection on the aerobic metabolism of Biomphalaria straminea and Biomphalaria tenagophila (Mollusca, Gastropoda)[END_REF]. The activation of anaerobic metabolism (LDH), enables continued production of energy (ATP), avoids inhibition of the glycolysis pathway by increased pyruvate levels, facilitates availability of NAD+ (oxidized electron carrier), and supports formation of intermediate metabolites that are needed for the survival of the host and for parasite development [START_REF] Lima | The influence of Angiostrongylus cantonensis (Nematoda, Metastrongylidae) infection on the aerobic metabolism of Biomphalaria straminea and Biomphalaria tenagophila (Mollusca, Gastropoda)[END_REF].

Ultimately, prolonged infection (especially with high parasite load), leads to depleted carbohydrate deposits that cause the snail metabolism to use alternative substrates as energy source, leading to breakdown of lipids (Tunholi et al., 2011a) and proteins [START_REF] Tunholi-Alves | Activation of anaerobic metabolism in Biomphalaria glabrata (Mollusca:Gastropoda) experimentally infected by Angiostrongylus cantonensis (Nematoda, Metastrongylidae) by high-performance liquid chromatography[END_REF]. Direct absorption of amino acids by developing larval A. cantonensis may further reduce protein levels; higher infection loads led to increasingly lower protein concentrations in A. fulica [START_REF] Tunholi-Alves | Unveiling the oxidative metabolism of Achatina fulica (Mollusca:Gastropoda) experimentally infected to Angiostrongylus cantonensis (Nematoda: Metastrongylidae)[END_REF]. The carbon backbone of proteins supplies intermediate compounds for neoglucogenesis but continued protein degradation not only reduces protein concentration but also leads to elevated levels of nitrogen compounds, altering the chemical composition of snail host hemolymph [START_REF] Pinheiro | Physiological changes in Lymnaea columella (Say, 1818) (Mollusca, gastropoda) in response to Echinostoma paraensei Lie and Basch, 1967 (Trematoda, Echinostomatidae) infection[END_REF]. The excretion as urea, uric acid or ammonia, of amino groups that result from deamination, challenge the water balance of A. fulica, a terrestrial gastropod [START_REF] Tunholi-Alves | Activation of anaerobic metabolism in Biomphalaria glabrata (Mollusca:Gastropoda) experimentally infected by Angiostrongylus cantonensis (Nematoda, Metastrongylidae) by high-performance liquid chromatography[END_REF]. While specific data from A. fulica are not available, Angiostrongylus infections may reduce the lifespan of snail hosts (Wallace and Rosen, 1969c).

Immunity of Achatina fulica

Despite considerable initial investigative attention from comparative immunologists, general aspects of the immune function in A. fulica are interpreted largely by considering findings from several other gastropod species, as presented in section 4.1. This is with the realization that some aspects of immune defense will be lineage-specific, however, the sharing of several immune features across animal evolution and the commonalities evident among immune defenses of phylogenetically diverse gastropods motivate reasonable confidence that this approach is generally informative. For example, a limited diversity of types of AMPs characterizes gastropods of both the Planorbidae and Physidae (sister families within the Hygrophila, Panpulmonata), and caenogastropods share particular categories of immune lectins with panpulmonate snails [START_REF] Loker | Invertebrate immune systems-not homogeneous, not simple, not well understood[END_REF][START_REF] Gorbushin | Lectin-like molecules in transcriptome of Littorina littorea hemocytes[END_REF][START_REF] Gorbushin | Derivatives of the lectin complement pathway in Lophotrochozoa[END_REF][START_REF] Schultz | Comparative immunological study of the snail Physella acuta (Hygrophila, Pulmonata) reveals shared and unique aspects of gastropod immunobiology[END_REF]. Likewise, the available insights specifically regarding A. fulica immunity (4.2) include antimicrobial proteins found throughout the gastropod clade, as well as a capacity for hemolymph coagulation not found in hygrophilid panpulmonates. It is of note that immunological studies of A. fulica have employed mostly cell biology and biochemical-type analyses that did not yield a detailed characterization of the factors involved as is now common in the "omics"-era. In light of resurging interest in A. fulica, also for the reasons provided in this review [START_REF] Penagos-Tabares | Novel approach to study gastropod-mediated innate immune reactions against metastrongyloid parasites[END_REF], such data may be anticipated in the near future.

General consideration of gastropod host-pathogen immune interactions

Historically, studies of gastropod immunity have largely focused toward immune interactions between aquatic vector snails and digenetic trematodes, such as the medically relevant schistosomes, with occasional consideration of other (microbial) pathogens [START_REF] Adema | Differential transcriptomic responses of Biomphalaria glabrata (Gastropoda, Mollusca) to bacteria and metazoan parasites, Schistosoma mansoni and Echinostoma paraensei (Digenea, Platyhelminthes)[END_REF][START_REF] Deleury | Specific versus non-specific immune responses in an invertebrate species evidenced by a comparative de novo sequencing study[END_REF][START_REF] Loker | Molluscan immunobiology: challenges in the Anthropocene Epoch[END_REF].

Snail immune systems, like those of other invertebrates [START_REF] Murthy | Invertebrates as model organisms for research on aging biology[END_REF] do not include vertebrate-type acquired immunology but comprise highly effective innate immune mechanisms. Immune function can be selective, allowing mutualistic interactions between gastropods and microbiota; commensal communities of bacteria and fungi can be instrumental in adaptation and evolution by affecting multiple fitness parameters of the host [START_REF] Soen | Environmental disruption of host-microbe co-adaptation as a potential driving force in evolution[END_REF], even viruses can modulate host-parasite interactions in invertebrates [START_REF] Galinier | A multistrain approach to studying the mechanisms underlying compatibility in the interaction between Biomphalaria glabrata and Schistosoma mansoni[END_REF]. Normally, however, (opportunistic) pathogens are countered by potent internal defenses. Actual immune-elimination by snails of metazoan parasites such as digenetic trematodes depends on complex interactions between determinants of compatibility. First, snail immune receptors must be able to recognize the antigens of the invader in order to activate host defenses. Intra-individual polymorphisms of such receptors and antigens determine matched versus mismatched status of host and parasite phenotypes, resulting in either survival or immune-elimination of the parasite [START_REF] Mitta | The Compatibility between Biomphalaria glabrata snails and Schistosoma mansoni[END_REF]. Secondly, host immune systems may be negated by anti-effector strategies of the parasite [START_REF] Lie | Swellengrebel lecture: survival of Schistosoma mansoni and other trematode larvae in the snail Biomphalaria glabrata. A discussion of the interference theory[END_REF].

These interactions play out in a continuous arms race that drives evolution of (mechanisms that generate) diversity and polymorphism of the factors that determine the outcome of the host-parasite interplay [START_REF] Combes | Selective pressure in host-parasite systems[END_REF][START_REF] Gourbal | Polymorphic mucin-like protein in Schistosoma mansoni, a variabale antigen and a key component of the compatibility between the schistosome and its snail host[END_REF]. For instance, antigenic variation is a common strategy of eukaryote pathogens to evade a host immune response [START_REF] Finlay | Anti-immunology: evasion of the host immune system by bacterial and viral pathogens[END_REF][START_REF] Perrin | Schistosoma mansoni mucin gene (SmPoMuc) expression: epigenetic control to shape adaptation to a new host[END_REF].

Absent the vertebrate-specific rearranging genes that yield specific immune receptor mechanisms (Di Noia and Neuberger, 2007; [START_REF] Tonegawa | Somatic generation of antibody diversity[END_REF], the remarkable effectiveness of invertebrate immune function is underscored by the evolutionary success of invertebrates [START_REF] Pinaud | A shift from cellular to humoral response contributes to innate immune memory in vector snail Biomphalaria glabrata[END_REF]. Evidence has mounted that also invertebrates, and gastropods specifically, are capable of considerable diversification of innate-type immune receptors and possess forms of immune memory to effectively counter diverse pathogens [START_REF] Zhang | Diversification of Ig superfamily genes in an invertebrate[END_REF][START_REF] Ghosh | Invertebrate immune diversity[END_REF][START_REF] Müller | An evolutionary perspective on the systems of adaptive immunity[END_REF][START_REF] Netea | Trained immunity: a memory for innate host defense[END_REF][START_REF] Wang | The response of mRNA expression upon secondary challenge with Vibrio anguillarum suggests the involvement of C-lectins in the immune priming of scallop Chlamys farreri[END_REF][START_REF] Pinaud | A shift from cellular to humoral response contributes to innate immune memory in vector snail Biomphalaria glabrata[END_REF].

Snail immune defenses include the body surface as external physical barriers, chemically fortified by (mucus) secretions containing antimicrobial factors. Pathogens that penetrate through these defenses encounter lytic factors, antimicrobial proteins and peptides (AMPs) in tissues and hemolymph. The antimicrobial bacterial pathogenassociated molecular patterns (PAMPs; also referred to as microbe-associated molecular patterns: MAMPs) or damage-associated molecular patterns (DAMPs) are recognized by pattern recognition receptors (PRRs). The PRRs are highly diverse, comprising secreted molecules, cell surface-and cytoplasmic receptors [START_REF] Schmitt | Immune responses in the Pacific oyster Crassostrea gigas. An overview with focus on summer mortalities[END_REF][START_REF] Escoubas | Immunity in molluscs[END_REF]. Among soluble receptors are Gram-negative bacteriabinding protein (GNBP), peptidoglycan recognition protein (PGRP) and a variety of lectins, including fibrinogen-related proteins (FREPs) [START_REF] Escoubas | Immunity in molluscs[END_REF][START_REF] Gorbushin | Derivatives of the lectin complement pathway in Lophotrochozoa[END_REF]. Cell surface receptors like Toll-like receptors (TLRs) and scavengers receptors (SRs) are highly conserved evolutionarily [START_REF] Peiser | Scavenger receptors in innate immunity[END_REF][START_REF] Sarrias | The scavenger receptor cysteinerich (SRCR) domain: an ancient and highly conserved protein module of the innate immune system[END_REF], just like cytoplasmic receptors from the RIG-I-like helicase (RLH) family and nucleotide-binding oligomerization domain (NOD)-like receptors [START_REF] Sirrad | Nod-like receptors: cytosolic watchdogs for immunity against pathogens[END_REF][START_REF] Yoneyama | RIG-I family RNA helicases: cytoplasmic sensor for antiviral innate immunity[END_REF][START_REF] Barber | Innate immune DNA sensing pathways: STING, AIMII and the regulation of interferon production and inflammatory responses[END_REF]. PPRbinding leads to coagulation, (direct) opsonization, triggering of proteolytic enzyme cascades that produce toxic compounds, or initiates intracellular signaling to activate immune defense cells to exert cytotoxicity (van der [START_REF] Van Der Knaap | Cytophilic receptors for foreignness and some factors which influence phagocytosis by invertebrate leucocytes: in vitro phagocytosis by amoebocytes of the snail Lymnaea stagnalis[END_REF][START_REF] Janeway | The role of microbial pattern recognition in self: non-self-discrimination in innate and adaptative immunity[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] Hanington | Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection[END_REF][START_REF] Clec'h | Characterization of hemolymph phenoloxidase activity in two Biomphalaria snail species and impact of Schistosoma mansoni infection[END_REF].

The cellular mediators of gastropod immunity are circulating macrophage-like phagocytic immune cells, named hemocytes, capable of production of cytolytic molecules including reactive intermediates of oxygen and nitrogen, as well as of phagocytosis and encapsulation of pathogens (Levine and [START_REF] Strand | Insect hemocytes and their role in immunity[END_REF][START_REF] Zanker | Immunology of Invertebrates: Humoral[END_REF]. Hemocytes are the main effectors of the defense system but they are also involved in wound repair, nutrient transport, shell calcification, digestion and excretion processes (Cheng, 1996). Based on morphology, hemocytes are commonly classified into three main cell types: granulocytes, hyalinocytes and blast-like cells [START_REF] Allam | Immune responses to infectious diseases in bivalves[END_REF][START_REF] Mahilini | Categorization of hemocytes of three gastropod species Trachea vittata (Muller), Pila globosa (Swainson) and Indoplanorbis exustus (Dehays)[END_REF][START_REF] Cavalcanti | Morphological characterization of hemocytes from Biomphalaria glabrata and Biomphalaria straminea[END_REF]. Hemocytes bear yet additional surface receptors like integrins, SRs, and C-type lectins [START_REF] Lv | The emergence of angiostrongyliasis in the People's Republic of China: the interplay between invasive snails, climate change and transmission dynamics[END_REF][START_REF] Hanington | Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection[END_REF] and will internalize pathogens (bacteria, fungi, and experimental particles like latex beads) through phagocytosis, larger targets such as larval helminth are encapsulated by multiple hemocytes. Little is known about the molecular processes involved in encapsulation [START_REF] Ray | Density shift, morphological damage, lysosomal fragility and apoptosis of hemocytes of Indian molluscs exposed to pyrethroid pesticides[END_REF][START_REF] Queiroga | Immunological responses of the mangrove oysters Crassostrea gasar naturally infected by Perkinsus sp. in the Mamanguape Estuary, Paraiba state (Northeastern, Brazil)[END_REF][START_REF] Francisco | Parasites and symbionts from Mytilus galloprovincialis (Lamark, 1819) (bivalves: Mytilidae) of the Aveiro Estuary Portugal[END_REF][START_REF] Laruelle | Histological analysis of trematodes in Dreissena polymorpha: their location, pathogenicity, and distinguishing morphological characteristics[END_REF][START_REF] Mladineo | Reaction of the mussel Mytilus galloprovincialis (Bivalvia) to Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria) concurrent infestation[END_REF][START_REF] Batista | Infestation of the cupped oysters Crassostrea angulata, C. gigas and their first-generation hybrids by the copepod Myicola ostreae: differences in susceptibility and host response[END_REF][START_REF] Kawasaki | Hemiuroid trematode sporocysts are undetected by hemocytes of their intermediate host, the ark cockle Anadara trapezia: potential role of surface carbohydrates in successful parasitism[END_REF][START_REF] Wootton | Interaction between non-specific electrostatic forces and humoral factors in haemocyte attachment and encapsulation in the edible cockle, Cerastoderma edule[END_REF][START_REF] Wang | A multi-CRD C-type lectin with broad recognition spectrum and cellular adhesion from Argopecten irradians[END_REF].

Efficiency of cellular immune defense, and perhaps also the ability to effectively counter parasites may increase with the intrinsic number of circulating hemocytes [START_REF] Larson | Resistance of Biomphalaria glabrata 13-16R1 snails to Schistosoma mansoni PR1 is a function of haemocyte abundance and constitutive levels of specific transcripts in haemocytes[END_REF]. The number of hemocytes in hemolymph varies between and within species [START_REF] Adema | The effect of homolymph extraction on distribution of lysosomal enzymes in Lymnea stagnalis: a cytochemical study[END_REF], because of age, infection and under influence of physiological and environmental factors [START_REF] Livingstone | Carbohydrate Metablismo of gastropods, p. 177242[END_REF]. For the actual killing of pathogens, hemocytes produce and release lysosomal enzymes, antimicrobial peptides, reactive species hydrogen peroxidase, ROS and RNS [START_REF] Loker | Gastropod immunobiology[END_REF][START_REF] Schmitt | Immune responses in the Pacific oyster Crassostrea gigas. An overview with focus on summer mortalities[END_REF]. Continued research may broaden the perspective of general immune defenses, for instance an ancient and evolutionarily conserved host defense mechanism was revealed by the recent finding that animal immune cells may deploy their DNA as extracellular traps (ETs) to capture pathogens [START_REF] Poirier | Antimicrobial histones and DNA traps in invertebrate immunity: evidences in Crassostrea gigas[END_REF]Robb et al., 2014). Relatively little is known about antinematode immunity in gastropods, however, literature indicates that snail species do not indiscriminately support the development of any nematode species that they may encounter [START_REF] Dróźdź | Étude des morphologie et evolution larvaires de Angiostrongylus (Parastrongylus) dujardini Dróźdź et Doby, 1970 Nematoda: Metastrongyloidea. Infestation des mollusques hôtes intermédiares[END_REF], however, several species of nematodes attract but most often do not appear to be eliminated by snail host immunity [START_REF] Bayne | Molluscan immunobiology[END_REF]. Further investigations may help clarify to what extent immune function is a codeterminant of vector suitability of a snail like A. fulica for A. cantonensis.

Immune features of Achatina fulica

The following aspects of immunity have been shown specifically from A. fulica. Included are immune features that are distributed across a wide phylogenetic range of gastropods, like achacin representing a category of conserved antimicrobial proteins and that are more restricted like ability for hemolymph coagulation, that has not been observed from e.g. aquatic panpulmonate gastropods.

Shell encapsulation of nematodes

Exploration of the use of nematode infection as control measure against snail pests revealed that A. fulica was remarkably resistant to the parasitic nematode Phasmarhabditis hermaphrodita, a lethal pathogen that is effectively against several other gastropod species. Starting at 3 days post exposure, inspection of experimental snails evidenced an immune response that involved the A. fulica shell. Infective P. hermaphrodita larvae are encapsulated, killed and remain permanently encased in the shell (Williams andRae, 2015, 2016). This immune mechanism is effected by cells on the inner layer of the shell that adhere to the nematode cuticle, spread over the worm body to then fuse and encase the nematode to the inside of the shell. Remarkably, DNA extracted from shell-encased dead worms allows for PCR and sequence-based identification of the infecting nematodes. Follow-up investigation with experimentally exposed and field collected snails showed that this novel anti-nematode immune mechanism is shared by snail species of both achatinoid and non-achatinoid clades of the Stylommatophora [START_REF] Rae | The gastropod shell has been co-opted to kill parasitic nematodes[END_REF].

Achacin

In 1985, Kubota et al., isolated a protein with an antibacterial activity, composed of 70-80 kDa subunits from the body surface mucus of A. fulica. Further characterization of this protein designated as Achacin yielded the first cDNA sequence from A. fulica ever deposited in GenBank [START_REF] Obara | Molecular cloning of the antibacterial protein of the giant African snail, Achatina fulica Férussac[END_REF]. Achacin (purified or recombinantly expressed) inhibited growth of Gram-negative and Gram-positive bacteria, and it was shown to attack the surface membrane of bacteria but not directly cause lysis bacterial cells [START_REF] Otsuka-Fuchino | Bactericidal action of a glycoprotein from the body surface mucus of giant African snail[END_REF], 1993;[START_REF] Ogawa | Macromolecular antimicrobial glycoprotein, achacin, expressed in a methylotrophic yeast Pichia pastoris[END_REF]. The mode of antibacterial action of Achacinin was first indicated by similarities in amino acid sequence with Lamino acid oxidases (LAAOs) from toxins in snake venoms and experimentally demonstrated to result from oxidative deamination of amino acids, producing ketoacids, ammonia (NH3) and hydrogen peroxide (H2O2). Additional to generating toxic metabolites, also depletion of amino acids leading to reduction of available nutrients was proposed to contribute to the microbicidal activities of Achacin [START_REF] Ehara | Antimicrobial action of achacin is mediated by Lamino acid oxidase activity[END_REF]. It is of note that Achacin presents a landmark discovery of comparative immunology of gastropods and molluscs. Starting with a report of Aplysianin from Aplysia kurodai (sea hare) an euopisthobranch gastropoda [START_REF] Kamiya | Aplysianin-A, an antibacterial and antineoplasmic glycoprotein in the albumen gland of a sea hare, Aplysia kurodai[END_REF][START_REF] Takamatsu | Molecular cloning of the defense factor in the albumen gland of the sea hare Aplysia kurodai[END_REF], many additional LAAOs been recovered from several gastropod and other molluscs. Phylogenetic analyses distinguish molluscan LAAOs as a major subfamily separate from other animal LAAOs, such independent evolution indicates an important reliance of molluscs on LAOOs for innate immune function [START_REF] Hughes | Origin and diversification of the L-amino oxidase family in innate immune defenses of animals[END_REF][START_REF] Suwannapan | Amplification and bioinformatics analysis of conserved FADbinding region of L-amino acid oxidase (LAAO) genes in gastropods compared to other organisms[END_REF].

Mytimacin and other cytoloytic peptides from mucus

The body surface mucus of A. fulica contains multiple components with lytic activity additional to Achacin. [START_REF] Zhong | A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica[END_REF] purified Mytimacin-AF, a cysteine-rich antimicrobial peptide (AMP) from the macin family. The cDNA sequence contains an open reading frame that encodes for an 80 amino acid-long peptide that has antimicrobial activity against fungi, Gram-negative and Gram-positive bacteria, particularly Staphylococcus aureus. A search for potential anticancer peptides motivated a broad survey of novel lytic factors present in A. fulica mucus. Bioinformatics analyses of mass spectrometry data obtained from two HPLC-fractions of mucus that displayed lytic activity toward in vitro cultured primate epithelial cells and (human) cancer cells, predicted another 16 lytic peptides in silico [START_REF] E-Kobon | Prediction of anticancer peptides against MCF-7 breast cancer cells from the peptidomes of Achatina fulica mucus fractions[END_REF]. The lack of sequence similarity of predicted lytic peptides with existing entries in public sequence databases, suggest that these mucus-derived lytic peptides have not been encountered previously, but was also considered to reflect the limited availability of sequence data from mollusc, especially the stylommatophoran land snails, especially (EKobon et al., 2015).

Lectins

Considering current insights into the lectin diversity among gastropods (e.g [START_REF] Gorbushin | Lectin-like molecules in transcriptome of Littorina littorea hemocytes[END_REF], it was not surprising that initial searches for agglutinins as mediators of non-self recognition in A. fulica led to identification of several (mucus and) hemolymph lectins, each with different carbohydrate specificity [START_REF] Basu | A single step purification of a sialic acid binding lectin (AchatininH) from Achatina fulica snail[END_REF]Mitra and Sarkar, 1988;[START_REF] Sarkar | A new cold agglutinin from Achatina fulica snails[END_REF]Mitra et al., 1988). Relative levels of lectin-mediated hemagglutinating activity increase with age of A. fulica snails (Baskakov et al., 2000), indicating a maturation of immune function from juvenile to adult A. fulica. Befitting the technological capabilities at the time of these pioneering studies, these lectins were identified only by carbohydrate specificity and the molecular weight of biochemically purified (subunits of) proteins, sequence characterization has remained pending, to date. Functional aspects, however, have been studied in greater detail for two lectins.

The first is an abundant hemolymph lectin of A. fulica, designated as C-reactive protein (CRP) based on crossreactivity with antisera to CRP protein of the horse shoe crab Limulus polyphemus (Agrawal et al., 1990) and specific binding of phosphoryl choline, a trait shared with CRPs (members of the pentraxins), innate immune proteins from both invertebrate and vertebrate animals, [START_REF] Mandal | Isolation of a phosphoryl choline-binding protein from the hemolymph of the snail, Achatina fulica[END_REF]. The amino acid content was determined and A. fulica CRP (a multimeric protein of 400 kDa) was found to be heavily glycosylated (30-40% carbohydate), but no nuclueotide sequence is currently available. Agglutinating activity was enhanced in the presence of calcium-ions [START_REF] Mandal | Isolation of a phosphoryl choline-binding protein from the hemolymph of the snail, Achatina fulica[END_REF]. The CRP of A. fulica is multifunctional, with bacteriostatic activity for Gram (-) bacteria and bactericidal activity for Gram (+) bacteria, likely through ROS production and induction of apoptosis, and it may serve to protect the snail against toxicity from heavy metals [START_REF] Mukherjee | Ancient origins of vertebratespecific innate antiviral immunity[END_REF][START_REF] Bose | C-reactive protein in the hemolymph of Achatina fulica: interrelationship with sex steroids and metallothionein[END_REF].

Secondly, research has also focused on Achatinin [START_REF] Basu | A single step purification of a sialic acid binding lectin (AchatininH) from Achatina fulica snail[END_REF]. This (9-O-AcSA)-specific hemolymph lectin (242 kDa, subunits of 15 kDa) is expressed in the albumen gland [START_REF] Sen | Albumen gland of the snail Achatina fulica is the site for synthesis of AchatininH, a sialic acid binding lectin[END_REF] and in hemocytes [START_REF] Biswas | Investigation on interaction of Achatinin, a 9-Oacetyl sialic acid-binding lectin, with lipopolysaccharide in the innate immunity of Achatina fulica snails[END_REF], binds sialic acid-epitopes on LPS in a calcium-dependent manner and is bacteriostatic toward Gram-negative Escherichia coli. Remarkably, Achatinin is also multifunctional, additional to antimicrobial activity, LPS binding of this PRR also activates other aspects of A. fulica immunity as is described in the next section.

LPS-mediated coagulation

Bacterial LPS activates a defense response in A. fulica hemocytes that involves the release of several interactive proteins into the hemolymph that orchestrate a coagulation reaction; gel formation to aggregate and sequester bacteria. This response is highly reminiscent of well-characterized endotoxin-dependent plasma coagulation from the horseshoe crab Limulus polyphemus, resulting from an enzyme cascade that employs zymogens and serine proteases released by amboecytes that degranuate in response to detection of LPS.

The coagulation reaction of A. fulica can be affected in vitro with the protein lysate of snail hemocytes [START_REF] Biswas | The role of amoebocytes in the endotoxin-mediated coagulation in the innate immunity of Achatina fulica snail[END_REF]. The name of this preparation, "Achatina amboecyte lysate (AAL)" may seem less intuitive because it employs the alternative designation of amoebocyte for hemocyte. Protein biochemical analysis implicated Achatinin as major, important component of AAL. Depletion of Achatinin reduced the coagulation activity of AAL by 77%. Additional to lectin function (LPS binding), Achatinin also has enzymatic serine protease activity that is critical for the coagulation, as shown by sensitivity of this reaction to protease inhibitors [START_REF] Biswas | Investigation on interaction of Achatinin, a 9-Oacetyl sialic acid-binding lectin, with lipopolysaccharide in the innate immunity of Achatina fulica snails[END_REF].

Another protein component, endotoxin-senstive factor (ESF), was purified from AAL using heparin affinity chromatography. ESF differs from Achatinin by a native molecular weight of 140 kDa (consisting of two non-covalenty bound 70 kDa subunits), and although both of these immune factors bind LPS, ESF is activated by alpha-chymotrypsin instead of endotoxin. Again, the use of specific inhibitors showed that active ESF functions a serine protease [START_REF] Biswas | Investigation on interaction of Achatinin, a 9-Oacetyl sialic acid-binding lectin, with lipopolysaccharide in the innate immunity of Achatina fulica snails[END_REF]. It is of note that the endotoxin-dependent coagulation reaction of A.fulica may be unique to Achatinoid gastropods (e.g. Archachatina marginata, [START_REF] Salawu | Effect of Escherichia coli endotoxin on Archachatina marginata hemolymph coagulation system[END_REF] or to (larger taxonomic subsets of) the Stylommatophora; it has not been reported fresh water pulmonate snails (Hygophila) like the well studied B. glabrata a main model for gastropod immunity. Along with characterization of all AAL factors and the mechanism of the protein cascade needed for coagulation, the phylogenetic distribution of this immate immune response merits further investigation.

Hemocytes

The hemolymph of A. fulica can easily be sampled by heart puncture, a procedure that can be survived by the snail. Microscopical observation delineated two categories of cell types among glass-adherent hemocytes; granulocytes and agranulocytes [START_REF] Adema | The effect of homolymph extraction on distribution of lysosomal enzymes in Lymnea stagnalis: a cytochemical study[END_REF], generally corresponding with hemocyte types recognized in other gastropods [START_REF] Yoshino | The ultrastucture of the circulating hemoymph cell of the marine snail Cerithidea californica (Gastropoda: Prosobranchia)[END_REF][START_REF] Barraco | Morphological characterization of the hemocytes of the pulmonate snail Biomphalaria tenagophyla[END_REF]. [START_REF] Karuthapandi | Studies on the hemocytes of Achatina fulica[END_REF] revealed that the total hemocyte count of active A. fulica snails was higher than that of the snails that aestivated. The granulocytes of A. fulica sub categorized into granulocyte I, granulocyte II and granulocyte III. Only few round small cells with high nucleus to cytoplasm ratio were observed. Overall, agranulocytes and granulocytes were proportioned at an 80%/20% ratio in the hemolymph of A. fulica, without evidence of sesonal variation. Differential hemocyte counts revealed only modest variation in the percentage of agranulocytes, granulocyte I and granulocyte III of active versus aestivated snails, whereas the percentage of granulocyte II was higher in aestivated A. fulica relative to active snails [START_REF] Karuthapandi | Studies on the hemocytes of Achatina fulica[END_REF], perhaps due to the difference in metabolic state, especially food uptake.

Basic immune functions of A. fulica hemocytes as snail defense cells were demonstrated in vitro by phagocytosis of zymosan particles and production of ROS [START_REF] Adema | The effect of homolymph extraction on distribution of lysosomal enzymes in Lymnea stagnalis: a cytochemical study[END_REF]. More recently, an in vitro coculturing assay that was designed to investigate the cellular immune interactions of stylommatophoran snails with A. cantonensis shows that A. fulica hemocytes attach to nematode larvae and proceed to hemocyte clusters around the parasites [START_REF] Penagos-Tabares | Novel approach to study gastropod-mediated innate immune reactions against metastrongyloid parasites[END_REF]. The same study and work from [START_REF] Lange | Gastropod-derived haemocyte extracellular traps entrap metastrongyloid larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior[END_REF] also observed that hemocytes from A. fulica, as well as Arion lusitanicus and Limax maximus (gastropod species commonly referred to as slugs) deploy DNA-rich extracellular aggregates referred to as invertebrate extracellular phagocyte traps (InEPTs) similar to the phagocytederived extracellular traps (ETs) described from vertebrates catching larvae and decreasing their motility. It was proposed that hemocyte InEPTosis immobilizes nematodes to hamper migration of the larvae through the tegument or body and further facilitate exposure of the entrapped parasites to cell-mediated cytotoxicity of additional hemocytes. The long-term fate of the parasites remains to be determined.

Achatina fulica, entry into the omics era

As described above, study of A. fulica has not only provided some of the earliest insights into general aspects of gastropod immunity but also continues to contribute to a more comprehensive overview of innate immunity in invertebrates. A continued reliance, mostly on cellular and biochemical research approaches has provided a steady yet rather slow progress in characterization of immune function of A. fulica, and of stylommatophoran snails as a larger, important group within the Gastropoda. The lack of specific sequence data to define the factors that enable the fascinating immune processes recorded from A. fulica hinders comparative approaches that can reveal the phylogenetic distribution of unique and shared defense capabilities across gastropods. Increasingly, routine and accessible next generation sequencing (NGS) technologies are now available to explore the genome sequences and transcriptomes from non-model organisms. While it remains challenging to derive high quality assemblies, extensive genome sequence and RNAseq data can be analyzed using computational bioinformatics for (potential) immune genes to indicate presence of immune mechanisms within an organism relative to the phylogenetic position. The validity of this approach [START_REF] Dheilly | No more non-model species: the promise of next generation sequencing for comparative immunology[END_REF][START_REF] Schultz | Comparative immunogenomics of molluscs[END_REF] is underscored by a broader view of immune function in a specific gastropod like B. glabrata [START_REF] Adema | Whole genome analysis of a schistosomiasis-transmitting freshwater snail[END_REF] and the modeling of shared lectinbased complement-like system that serves immunity across the Mollusca and other protostome invertebrates [START_REF] Gorbushin | Derivatives of the lectin complement pathway in Lophotrochozoa[END_REF]. Molecular level approaches have more usually been applied for population biology (e.g. [START_REF] Ayyagari | Evaluation of haplotype diversity of Achatina fulica (Lissachatina) [Bowdich] from Indian sub-continent by means of 16S rDNA sequence and its phylogenetic relationships with other global populations[END_REF][START_REF] Fontanilla | Restricted genetic variation in populations of Achatina (Lissachatina) fulica outside of East Africa and the Indian Ocean Islands points to the Indian Ocean Islands as the earliest known common source[END_REF], but recently, the genome of A. fulica has become accessible with the publication of both the 15,057bp mitochondrial genome et al., 2016) and the nuclear genome (GuoY et al., 2019) the first to represent the stylommatophoran gastropods. The genome (estimated at 2.12 Gb) of a single A. fulica snail from the Guangxi region of China was sequenced to yield a publicly available high quality, chromosomal-level genome assembly of 1.85 Gb with a contig N50 length of 726 kb. The predicton of 23,726 protein-coding genes will be of great value to facilitate proteomics level studies. It is anticipated here that this significant data resource will lead to sequence characterization of immune factors previously identified from A. fulica through biochemical parameters only, like Achatinin, ESF and others, also providing provide a wideranging insight into related and additional immune genes in this snail species.

Conclusions and future prospects

By phylogenetic identity alone, A. fulica merits investigative attention due to the potential as representative of a major branch of the Stylommatophora, to inform broadly on gastropod immune function. Already, A. fulica research has indicated both unique-and fundamental, evolutionary conserved aspects of snail immunity. The great biological and evolutionary diversity within the class Gastropoda of the phylum Mollusca makes it highly probable that particular clades of snails have evolved unique immune features. Importantly, broadening studies beyond the main focus on a very small segment of gastropod diversity indicated the possibility of differences in the elaboration of immune function between gastropod families. Schultz and Adema, 2017 observed that Physella acuta (Physidae, sister family of the Planorbidae) snails did not equally employ FREPs, a category of PPRs that is instrumental for coordinating immune elimination of schistosome parasites in the planorbid snail B. glabrata, vector of schistosomiasis [START_REF] Hanington | Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection[END_REF].

Renewed interests to study A. fulica are driven by the recognition of multiple negative biological properties that render this snail a highly effective invasive species that is recalcitrant toward eradication, a pest that can destroy (crop)vegetation and outcompete native species, and a potential vector that may transmit several parasites including A. cantonensis causative nematode pathogen for eosinophilic meningitis, an emerging infectious disease in humans. Elucidation of the underlying traits that determine the range if negative biological impact opens possibilities to develop strategies for control of A. fulica, or to mitigate, modify, or at least predict the distribution of the snail and associated parasite transmission.

A modest but strong tradition of biochemically centered investigations provides a several entry points for more detailed characterization of A. fulica immunity, with great potential to benefit from the recently available genome sequence data. We thus strongly encourage further immunological studies of the invasive nature of A. fulica, and interaction of A. fulica with A. cantonensis. In the process such studies will yield exciing new insights also in comparative immunology of terrestrial snails to reveal evolutionary patterns in the evolution of immune function of Gastropoda.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dci.2019.103579.


Dr Lise-Marie Lacroix
email: lmlacroi@insa-toulouse.fr

Dr Anca Meffre

Dr Christophe Gatel

Dr Pier-Francesco Fazzini

Dr Sébastien Lachaize

Prof Marc Respaud

Dr Bruno Chaudret

Nanoparticle Ripening : A Versatile Approach for the Size and Shape Control of Metallic Iron Nanoparticles

Keywords: Moessbauer spectroscopy, iron, magnetic properties, nanoparticles, seeded growth TOC

A novel approach for the synthesis of Fe(0) nanoparticles (NPs) with tunable sizes and shapes is reported. Ultrasmall Fe(0) NPs were reacted under mild conditions in the presence of a mixture of palmitic acid and amine ligands. These NPs acted not only as preformed seeds but also as an internal iron(II) source that was produced by the partial dissolution of the NPs by the acid. This fairly simple approach allows a strict separation between the nucleation and the growth steps. By changing the acid concentration, a fine tuning of the relative ratio between remaining Fe(0) seeds and iron(II) reservoir was achieved, giving access to both size (from 7 to 20 nm) and a shape (spheres, cubes or stars) control. The partial dissolution of the ultrasmall Fe(0) NPs into iron(II) source and the successive growth was further studied by using combined TEM and Mössbauer spectroscopy. The successive corrosion, coalescence, and ripening observed could be understood in the framework of an environment-dependent growth model.

INTRODUCTION

Magnetic nanoparticles (NPs) are objects of choice for a wide range of applications, [1] such as data storage, [2], [3] catalysis, [4] and biomedical ones. [START_REF] Bouchard | Proc. Natl. Acad. Sci[END_REF], [START_REF] Haun | [END_REF] Among the different properties sought, the potential overheating of the nanoparticles in presence of an alternate magnetic field received tremendous interest due to its implication on Magnetic Field Hyperthermia (MFH) treatment [7] and thermally-assisted catalysis. [8], [9] To benefit from optimized heating properties, high saturation magnetization (M S ) materials are mandatory. [10], [11] Metallic Fe NPs exhibit magnetization far above their oxide counterparts. However, due to the reactivity of iron towards oxidation, synthesis and stabilization of such fully metallic nanoobjects remains a challenge. [12] The thermodecomposition of Fe(CO) 5 in the presence of long-chain ligands has been used in several occasions. [13], [14] However, the magnetic properties of these NPs are usually degraded compared to bulk values due to synthesis by-products such as carbon and oxygen impurities. Recently, alternative precursors such as Fe(oleate) 3, [15] Fe(C 5 H 5 )(C 6 H 7 ), [16] or Fe{N[Si(CH 3 ) 3 ] 2 } 2 , [17] leads to NPs exhibiting high magnetization and narrow size distribution. Yet, the control of the nanoparticle size and shape, and thus the physical properties of the objects have scarcely been addressed. [18] In contrast to Co and CoNi alloy, for which anisotropic shapes could be reached by tailoring the adopted crystalline structure, [19], [20] such morphological control was not yet achieved for Fe, which exposes a body centered cubic structure. To reach the nanoscale design required for the fine tuning of both size and shape of Fe NPs, a deep understanding of the underlying nucleation and growth mechanisms is needed.

Following an organometallic approach, we were able to produce nanoparticles with a mean diameter ranging between 1.5 and 9 nm, [18] and single crystal nanocubes self-organized into micrometric superlattices by reduction of {Fe[N(SiMe 3 ) 2 ] 2 } 2 (Me = CH 3 ) in presence of long chain acid and amine under H 2 at 150°C. [17] Thanks to a detailed kinetic study and the characterization of the intermediate complexes formed in-situ, among which Fe carboxylate, we could propose a complex 3-step mechanism to rationalize the synthesis. First, as classically invoked, nucleation proceeded. Then, a growth occurred concomitantly to a coalescence step, in presence of an amine rich environment. Finally a growth-ripening step is observed, induced by the release of carboxylate. Playing with the experimental conditions one can favor any of these three steps. Lowering the reaction temperature or changing the amine/acid allowed favoring the coalescence, yielding stars [18] and porous cubes. [21] Replacing carboxylic acid by ammonium chloride salt changed the iron intermediates formed and thus fastened the reaction kinetic, yielding larger spheres and cubes. [22], [23], [24] To go beyond these results, we now need to gain control on the initial nucleation step. In the literature, the use of preformed seeds, in the so-called seeded-growth process, [25], [26] leads to the strict separation of the nucleation and the growth processes. Pt seeds have been successfully used to promote the heterogeneous growth of Fe NPs by thermodecomposition of Fe(CO) 5 . [13], [27] Recently, our group reported the overgrowth of Fe cubes onto preformed Co nanorods using {Fe[N(SiMe 3 ) 2 ] 2 } 2 . [28] Here, we propose a different approach where ultrasmall Fe(0) NPs of 1.5 nm [29] were used both as seeds and as Fe source, the idea behind consisting in taking advantage of the ripening step observed in the presence of carboxylic acid. The key role played by the hydrogen was evidenced by a combined transmission electron microscopy (TEM) and Mössbauer spectroscopy study.

RESULTS & DISCUSSION:

1.5 nm nanoparticles (NPs) were obtained by decomposing {Fe[N(SiMe 3 ) 2 ] 2 } 2 at 150°C under 3 bars of dihydrogen. [29] These ultrasmall particles were further used without any purification as reactive Fe(0) seeds. Ageing these seeds at 150°C with a mixture of ligands (palmitic acid, PA and hexadecylamine, HDA, Figure 1) under controlled atmosphere (H 2 or Ar) yield larger nanoparticles. Contrarily to the classical seeded-growth method used, no external iron source was added, the reactive Fe(0) seeds initially introduced being partly dissolved to form stable iron(II) species, they acted both as seeds and iron source, as further detailed below. The effect of the acid concentration on the aging process of Fe(0) seeds was studied at 150°C under H 2 atmosphere while keeping the amine concentration constant ([HDA] = 100mM). In presence of 50mM of palmitic acid, 7.0 ± 0.1 nm NPs were observed after 7 days of reaction at 150°C. Increasing the acid concentration to 75mM yielded slightly larger NPs exhibiting a mean diameter of 8.0 ± 0.1 nm NPs. In presence of an equi-proportion of acid and amine (100mM), the reaction was fastened as revealed by the important precipitation of 10.2 ± 0.1 nm NPs onto the magnetic stirrer after only 48h of ageing (Figure 2 andS1).

The final state of the ageing process, corresponding to the full reaction of the preformed Fe(0) seeds was never achieved. Indeed, unreacted seeds remained even after extended reaction time (up to 28 days) as characterized by TEM and zero-field cooling/field cooling magnetic measurements (Figure S2).

Large Fe(0) NPs could be easily retrieved from the reaction media using conventional centrifugation process. For instance, 10.2 nm NPs could be sorted and further characterized (Figure S3). The particles are highly crystalline and exhibit the expected bcc-Fe structure as revealed by X-Ray Diffraction (Figure 2e). Magnetic measurements performed at low temperature revealed a saturation magnetization of 215 Am²kg -1 , similar to the bulk value (Figure 2f). Mössbauer spectroscopy confirmed a pure Fe(0) composition, as revealed by the single sextet observed (Figure S3b).

Figure 2. Transmission electron microscopy (TEM) images of Fe NPs obtained from a) Fe(0) seeds after an aging process of (b-c) 7 days and d) 2 days in presence of 100mM of HDA and b) 50, c) 75 and d) 100 mM of PA. Scale bar corresponds to 50 nm. e) XRD pattern and f) Hysteresis cycle measured at 2K after a 5T field-cooling of the 10 nm spheres observed in d) after purification process. As inset : zoom of the +/-400 mT region, evidencing the absence of any exchange bias. The black lines in e) correspond to the bcc-Fe reference pattern (PDF-00-006-0696).

Starting from metallic Fe(0) seeds as new iron source for the synthesis of large NPs, the question of the role of H 2 , classically used as a strong reducing agent, naturally rose up.

Therefore a comparative study in absence of H 2 was performed. Fe(0) seeds were aged during 48h at 150°C under Ar in presence of an equi-concentration of PA and HDA (100mM). While 10.2 nm NPs were previously obtained under similar condition in H 2 atmosphere, large starlike shaped NPs were synthesized (Figure 3a). High resolution transmission electron microscope (HREM) revealed that these Fe NPs were single-crystalline but they do not exhibit any preferential exposed facets (Figure 3b). Similar ill-defined nanoparticles were previously observed as intermediate states prior to the stabilization of Fe nanocubes during the direct reduction of {Fe[N(SiMe 3 ) 2 ] 2 } 2 . [28] We invoked at that time an oriented-coalescence phenomenon in presence of an amine rich environment to explain the growth of such objects. Since we previously reported a successive ripening process which promoted the stabilization of {100} facets on Fe nanocubes, we tried to further extend the reaction time on our star-like shaped NPs up to 96h at 150°C under Ar.

However, the stars were fairly stable and no sign of shape evolution was detected (Figure S4).

On the contrary, allowing the star-like shaped NPs to react under H 2 at 150°C yielded facetted NPs (Figure 3c-d). HREM image and the corresponding Fourier transform (FT) confirmed that the facetted stars remained single-crystalline and clearly exhibit mostly {100} facets (Figure 3d andS5). The additional peaks appearing in the FT were due to the oxide shell formed after air exposure of the sample during the deposition of nanoparticles on the carbon foil and the insertion into the electron microscope.

Figure 3. a-c) TEM and b-d) HREM images of Fe NPs obtained in presence of 100 mM of PA and 100mM of HDA after a-b) a 48h aging process under Ar and c-d) after an additional 48h aging under H 2 . As inset : corresponding FT pattern confirming the bcc structure observed along [111] and [001] zone axis.

Playing with the ligand concentration, the size of the coalesced particles obtained after 48h of aging under Ar could be tuned between ca. 10 nm (50mM PA/ 100 mM HDA) and 25 nm (100mM PA/ 200mM HDA) (Figure S6). Once more, an additional ageing step under H 2 allowed for the faceting of these particles, leading to nanocubes and nanostars exhibiting {100} facets (Figure S7).

To go beyond morphological observations, a Mössbauer spectroscopy study was performed.

Figure 4 shows the evolution of Mössbauer spectroscopy patterns of Fe(0) seeds in presence of 100mM of PA and 100 mM of HDA as a function of the reaction time under either H 2 or Ar atmosphere. The spectra of the raw samples have been collected at low temperature 5K, to limit relaxation phenomena from superparamagnetic small Fe clusters. The analysis of the spectra was done on the basis of our previous Mössbauer investigations, for the seeds only [35] , and for the synthesis of NPs in presence of PA/HDA from the decomposition of {Fe[N(SiMe 3 ) 2 ] 2 } 2 . [18] Indeed, similar nano-objects and complexes are likely, as similar Fe precursor and ligands are used. All the spectra were then fitted with 4 contributions : Fe seeds [35] , ferromagnetic Fe, an extended phase named as [Fe] u_1 in [18], and molecular Fe(II) contributions.

[Fe] u_1 is not identified, but its high isomeric shift and the large hyperfine field are indicative of the presence of magnetically coupled Fe ions, in an acid environment. The different contributions are detailed in the supplementary information (Table S1, and Figure S8). At room temperature, the Fe(0) seeds quickly evolved, leading to the formation of a molecular complex iron(II) comparable to carboxylate species, as evidenced by the doublet observed at t = 0. [18] The seed corrosion was however not total, as revealed by the persistence of the seed signature. With time the molecular Fe(II) complex evolved to form the stable [Fe] u_1 species. In the following section the Fe(II) reservoir will refer to both Fe(II) molecular and [Fe] u_1 species.

Under H 2 , metallic Fe(0) species grew significantly but unreacted Fe(0) seeds along with Fe(II) reservoir remained after 48h (Table 1). The time evolution of the reaction was followed by a combined TEM study. Spheres grew homogeneously as revealed by TEM, their size increasing continuously from 2.8 ±0.2 nm after 30min to 7.1 ± 0.1 nm after 6h, 8.2 ± 0.1 nm after 12h and 9.9 ± 0.1 nm after 24h (Figure S9). The final size of 10.2 ± 0.1 nm was finally reached after 48h of reaction. Extended further the reaction time did not promote a further decomposition of the Fe(II) reservoir but rather lead to a size broadening. The complete reduction of the Fe(II) reservoir was also not achieved by direct decomposition of {Fe[N(SiMe 3 ) 2 ] 2 } 2 , [18] a competition being observed between the Fe(II) species reduction, which induced the liberation of acid species and the further corrosion of Fe(0) seeds.

Under Ar atmosphere, the corrosion of the Fe(0) seeds to give molecular complexes was also evidenced. After 3h, 60% of unreacted seeds still remained, while a small amount of Fe(0) NPs, not detected by Mössbauer were formed as revealed by TEM (Figure S10). Extending the reaction time up to 48h allows the stabilization of a larger proportion of Fe(II) reservoir, resulting from the seeds corrosion. The contribution of the star-like shaped Fe(0) NPs was limited to 16%, revealing that the reaction was far from being complete. Ultrasmall Fe(0) NPs were observed to be reactive at 150°C under H 2 in presence of a mixture of acid/amine ligands. An important proportion was corroded to form a fairly stable iron(II) reservoir which allows for the growth of the remaining unreacted seeds. Ultrasmall Fe(0) seeds acted thus not only as seeds but also as an internal iron source in this new approach. As expected the final size of the objects could be finely tuned by playing with the acid/amine ratio which governs the relative balance between the remaining seeds and the iron(II) reservoir. The higher the acid content, the larger the final NPs due to an extended iron(II) reservoir.

When the aging reaction was performed in presence of Ar atmosphere, the kinetic of both, the seed corrosion and the reduction of the iron(II) reservoir drastically slow down. Few star-like shaped nanoparticles were obtained, probably due to a coalescence process in an amine-rich like environment, as previously invoked for the direct synthesis of Fe NPs. [18] Thanks to a successive aging step in the presence of H 2 , NPs evolved to exhibit mainly (100) facets probably thanks to a growth-reparation mechanism when the acid molecules are slowly retrieved from the iron(II) reservoir, as previously proposed for the direct synthesis of Fe NPs. [18] CONCLUSION :

We report here a novel approach for the synthesis of metallic Fe nanoparticles, using ultrasmall NPs as seeds and iron source. In presence of acid/amine environment a complex ripening/coalescence occurs allowing a fine tuning of the final size and shape of the Fe(0) objects obtained. For the first time, star-like shaped Fe NPs exhibiting controlled facets could be reproducibly produced, opening the way for the stabilization of complex 3D magnetic structures. We believe that such simple approach could become a generic method for metallic NPs synthesis exhibiting complex shapes, indeed, preliminary studies have shown that it can be successfully transposed to Co(0) synthesis.

Experimental section :

Chemicals and Materials.

Due to the air sensitivity of the precursors and nanoparticles obtained, all synthesis were prepared under argon atmosphere by using Fischer-Porter bottles technique, glove box and argon/vacuum lines. Mesitylene (99% Fluka) and toluene (99%, VWR Prolabo), were distilled, degassed and transferred in the glove box prior to use. Hexadecylamine (HDA, 99%)

and palmitic acid (PA, 99%) were purchased from Sigma Aldrich and used without any additional purification. The bis(amido)iron dimer {Fe[N(SiMe 3 ) 2 ] 2 } 2 (Me = CH 3 ) was purchased from Nanomeps.

Synthesis of iron (0) nanoparticles

Step 1: Synthesis of preformed Fe(0) seeds.

Preformed Fe(0) seeds were prepared according to a previous procedure. [29] Briefly, in an Ar glove box, a green solution of {Fe[N(SiMe 3 ) 2 ] 2 } 2 (Me = CH 3 ) (0.5 mmol, 376 mg, corresponding to a Fe concentration of 50 mM) in 20 ml of mesitylene was transferred into a Fischer Porter bottle. The bottle was pressurized with 3 bars of dihydrogen for 10 minutes, and then transferred to a preheated oil bath at 150°C. The solution evolved quickly: after few minutes its color turned to black. The reaction was let to react 24h at 150°C under vigorous magnetic stirring, leading to a 20 mL black solution. After cooling down to room temperature, the reducing atmosphere was removed and the obtained solution was stored in the glove box.

Spherical nanoparticles of 1.5-2.0 nm in diameter homogeneous in size were quantitatively obtained.

Step 2: Ripening in presence of a mixture of ligands. Resolution-TEM (HRTEM) working with 100 kV and 200 kV respectively. In the majority of cases, size histograms were obtained by manual counting process over at least 100 particles, using ImageJ software. [30] Size distributions have been fitted by Gaussian law; the results are expressed by the calculated mean size and standard deviation σ. Magnetic studies were carried out on powder samples by SQuID (MPMS Quantum Design 5.5) and the iron state and environment were analysed by Mössbauer spectroscopy (WISSEL, 57 Co source). Samples were prepared into the glove box and extreme care was taken to avoid oxidation during transfer to the apparatus. Microanalysis measurements were performed by ICP (Inductively Coupled Plasma).

Figure 1 .

 1 Figure 1. Schematic view of the aging process of reactive Fe(0) seeds in presence of amine/acid ligand mixtures. Depending on the atmosphere, a growth or a complex coalescence/ripening can be observed.

Figure 4 .

 4 Figure 4. Mössbauer spectra measured at 5K of Fe seeds ripened in presence of 100 mM of PA and 100 mM of HDA at 150°C after different reaction time under Ar or H 2 .

  5mL of the obtained Fe(0) seed solution was used without any further purification and transferred into a Fisher Porter bottle. Palmitic acid (PA) and hexadecylamine (HDA) were added and carefully dissolved thanks to a strong magnetic stirring. The solution was then placed in an oil bath and allowed to react under 3 bars at 150°C for 2 to 7 days. * Synthesis of 10 nm spherical nanoparticles. Typically 128 mg of PA (0.5 mmol, 100mM) and 121 mg of hexadecylamine (0.5 mmol 100mM) were added to the 5mL seed solution. 10 nm NPs were obtained after 48h of reaction under H 2 at 150°C. Smaller NPs of 7 and 8 nm could be obtained by decreasing the acid content down to 64 mg (0.25 mmol, 50mM) and 96 mg (0.375 mmol, 75 mM) respectively, keeping an amine content of 0.5 mmol (100 mM).The reaction time was extended to 7 days under H 2 to counterbalance the decrease of reactivity observed. * Synthesis of star-like shaped particles. Typically 128 mg of PA (0.5 mmol, 100mM) and 121 mg of hexadecylamine (0.5 mmol, 100mM) were added to the 5mL seed solution. After 48h of reaction at 150°C under Ar, large coalesced NPs were obtained. The obtained solution was pressurized under 3 bars of H 2 and allowed to react further at 150°C for an additional 48h. Star-like shapes particles exhibiting controlled facets were finally obtained.Characterization of the NPs.Microscopy samples were prepared by deposition of a diluted colloidal solution drop on a carbon coated copper grid and observed on a JEOL-1011 for Bright Field Transmission Electronic Microscopy (TEM) and aberration corrected Tecnai F-20 microscope for High

Acknowledgment :

This work was supported by the French national project EMMA (ANR12 BS10 013 01) and the French microscopy network METSA.