Odhiambo Dickson

Anne Owuor
email: dowuor@strathmore.edu

Joseph Onderi Laurent

Olivier Orero
email: jorero@strathmore.edu

Lobry

Dickson Owuor
email: doowuor@lirmm.fr

Anne Laurent
email: anne.laurent@umontpellier.fr

Joseph Orero

Olivier Lobry
email: olivier.lobry@umontpellier.fr

Gradual Pattern Mining Tool on Cloud

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

Introduction

Scientific researchers are constantly collecting, crossing and analyzing data in order to help them understand various phenomena. For example environmental data is important for helping to understand phenomena like global warming, rainfall patterns etc. Most of such data is collected using sensors that are enabled to upload data in Cloud frameworks [START_REF] Liang | OGC SensorThings API part 1 : Sensing, version 1[END_REF][START_REF] Hajj-Hassan | Exploiting inter-and intra-base crossing with multi-mappings : Application to environmental data[END_REF][START_REF] Joshi | Sentiment analysis tool on cloud : Software as a service model[END_REF]. These researchers obviously wish to spend more time studying phenomena than configuring research tools. Fortunately, integrating data analysis tools into Cloud frameworks alleviates such configuration hustles.

It is important to highlight that understanding certain phenomena may require knowledge to be extracted from numerous unrelated data sets. The technique of combining unrelated data sets in order to identify interesting correlations, also known as data crossing, is increasingly gaining more interest in numerous research domains [START_REF] Hajj-Hassan | Exploiting inter-and intra-base crossing with multi-mappings : Application to environmental data[END_REF].

Temporal gradual pattern (GP) mining is an instance of a data analysis technique that allows for extraction of gradual correlations among attributes of a data set. For instance given a set of an ordered time-series data set with attributes {Temperature, Mosquitoes}, a temporal GP may take the form : "the higher the temperature, the more mosquitoes almost 2 hours later" [START_REF] Owuor | Mining fuzzy-temporal gradual patterns[END_REF]. Time-series data sets that are collected in real-time are also known as data streams. The OGC SensorThings API is an example of a framework that generates and stores data streams from numerous environmental sensors.

The idea of a software implementation that enables the access of GP mining algorithms through a SaaS (Software-as-a-service) model through a Web interface is interesting. In a SaaS distribution model, software is centrally hosted and licensed on a subscription basis. This introduces a flexibility that spares users the agony of spending hours trying to install analysis software [START_REF] Joshi | Sentiment analysis tool on cloud : Software as a service model[END_REF].

It is an added advantage if the software implementation allows users to select and cross numerous unrelated data streams into a single data set from which temporal GPs can be mined. For instance a user can cross a "temperature" data stream with a "no. of bees" data stream to test for a pattern like : "the higher the temperature, the higher the population of bees almost 3 days later".

According to [START_REF] Owuor | Mining fuzzy-temporal gradual patterns[END_REF] the computational complexity of mining GPs increases with the size of the data set. For this reason, the algorithms are implemented in Python language in order to harness Python's ability to improve efficiency through multi-core parallel processing. The proposed SaaS allows implementations of these algorithms on a powerful supercomputer and provides a user-friendly client interface for the users.

In this paper, we propose a software architecture model (on a Docker Cloud platform) that implements a crossing model for time-series data proposed by [START_REF] Owuor | Exploiting IoT data crossings for gradual pattern mining through parallel processing[END_REF]) and integrates T-GRAANK algorithm proposed by [START_REF] Owuor | Mining fuzzy-temporal gradual patterns[END_REF] in order to allow for extraction of GPs from crossed data streams. The remainder of this paper is organized as follows : we describe the OGC SensorThings API in Section 2 ; we describe the proposed model in Section 3 ; finally, we conclude and give future directions regarding this work in Section 4.

OGC SensorThings framework

(Open Geospatial Consortium) OGC1 is an international consortium consisting of over 530 companies, government agencies, research organizations and universities driven to make geospatial data and services FAIR (Findable, Accessible, Interoperable and Reusable). The OGC SensorThings API is an open standard that is built on top of the OGC Sensor Web Enablement (SWE) and (International Organization for Standardization) ISO/OGC Observation and Measurement (O&M) data model [START_REF] Van De Crommert | OGC web services in action[END_REF].

The SensorThings API is specifically designed for constrained IoT (Internet of Things) devices ; therefore, it employs the use of efficient technologies such as RESTful (Representational State Transfer) API, JSON (JavaScript Object Notation) encoding, MQTT protocol, OASIS OData (Open Data protocol) and flexible URL conventions [START_REF] Liang | OGC SensorThings API part 1 : Sensing, version 1[END_REF]. The OGC SensorThings API is composed of 2 parts : (1) the Sensing part and (2) the Tasking part. In this paper, we implement the Sensing part which allows IoT devices and applications to perform CRUD operations through HTTP requests (i.e. POST, GET, PATCH, DELETE).

The Sensing part is designed based on the ISO/OGC O&M data model and it defines 8 entities for IoT sensing applications. Figure 1 depicts the 8 entities together with their properties and relationships [START_REF] Hajj-Hassan | Integrating sensor data using sensor observation service : Towards a methodology for the o-life observatory[END_REF][START_REF] Liang | OGC SensorThings API part 1 : Sensing, version 1[END_REF]. When implemented, the OGC SensorThings exposes a service document resources, that lists the 8 entity sets, that allows its clients to navigate the entities in a hyper-media driven manner.

3 Proposed model

System architecture

We propose to extend GOST2 (golang SensorThings), a certified software implementation of the OGC SensorThings API, to allow for extraction of gradual patterns from unrelated data streams as illustrated in Figure 2.

We implement our model on top of the OGC SensorThings API because, to the best of our knowledge, it stands out as the best API to interconnect IoT devices, sensor data and applications over the Cloud. As can be deduced in Figure 2 As shown in Figure 2 and Figure 3, the proposed system is composed of 5 items which we described below.

(1) OGC SensorThings Service (implemented by GOST server) exposes the document resources of the 8 entity sets (listed in Section 2) for the SensorThings clients. This is the core task of the SensorThings API since every CRUD action go through it.

(2) Data Collection tool implements MQTT protocol which is a lightweight publishsubscribe protocol designed for constrained devices. This tool (implemented by Mosquitto server) is used to connect to sensors in order to collect data from the environment and send them to OGC SensorThings Service.

(3) Data Storage tool (implemented by GOST database) serves an object-relational database which implements the schema of the 8 OGC SensorThings sensing entities.

(4) Data Crossing tool serves a HTTP Dashboard which enables users to interact with our system. Through this tool users are able to send HTTP requests to the OGC SensorThings Service and the Pattern Mining tool. Specifically, this tool implements FuzzTX algorithm proposed by [START_REF] Owuor | Exploiting IoT data crossings for gradual pattern mining through parallel processing[END_REF] (5) Pattern Mining tool (implemented by GRAANK server) serves the GP mining algorithms which are variants of the T-GRAANK (Temporal GRAdual rANKing) technique proposed by [START_REF] Owuor | Mining fuzzy-temporal gradual patterns[END_REF] and are implemented in Python. This tool runs services that receive HTTP P OST requests from the Data Crossing tool and responds with the extracted patterns.

Demonstration

FIG. 4 -Screenshot of implemented Cloud application for demonstration

Our proposed Cloud application mostly targets users that require to cross unrelated timeseries data collected by sensors and mine them for patterns or knowledge that is not obvious. For demonstration purposes, we allow the application to mine for gradual patterns from crossed sensor data. We proceed to describe how users may interact with the system which is designed to run on a Web interface which is shown in Figure 4.

The left panel of the Web application provides navigation items for accessing : "Things", "Locations", "Historical Locations", "Sensors", "Observed Properties", "Datastreams", "Ob-servations", "Gradual Patterns" and "MQTT". The right panel provides an interface for any navigation item selected by the user. The user clicks on the "Things" navigation item to add a phenomena that he/she is interested in observing. The phenomena (or thing) allows the user to add define data streams, locations, and sensors that will collect data. The user can view these properties by clicking on "Datastreams", "Locations" and, "Sensors".

In order to cross different data streams and mine them for gradual patterns, the user clicks on the "Gradual Pattern" navigation item and the interface on the right panel will appear as shown in Figure 4. At the top of this panel, there are two configuration settings :

-first one allows the user to choose which data streams to cross and, -second one allows the user to specify the type and quality of the gradual patterns to be extracted. In this demonstration, the user selects "smoke detector" data stream and "oven temperature" data stream and specifies extraction of gradual patterns whose support is higher than 0.5. Below the configuration settings, is the results interface and it displays two complementary patterns that give the same knowledge. The gradual pattern can be interpreted as "the higher the oven temperature, the less the smoke" or vice-versa. We point out that the data streams used to mine this pattern are synthetic for the purposes of demonstration and they do not reflect any real scenario.

Possible application area

OREME (Observatory of Mediterranean Research of the Environment) is a scientific observatory that complies with INSPIRE3 Implementing Rules and supports research activities that involve continuous observation of nature over long periods in the Mediterranean region.

Currently, OREME provides OGC geospatial standards like WFS (Web Feature Service) and WMS (Web Map Service) through its data portal4 ; however, it has not yet implemented data stream standards like the OGC SensorThings due to data entity incompatibility. One solution may involve installing IoT sensors at the data collection stages, which integrate into an MQTT instance to generate compatible data streams [START_REF] Grellet | Workshop : Integration of O&M data in the INSPIRE SDI -Benefits, challenges and prospects[END_REF][START_REF] Kotsev | Extending INSPIRE to the Internet of Things through Sen-sorThings api[END_REF].

Conclusions and future works

In this paper, we propose and develop a software architecture model that allows gradual pattern mining algorithms to be accessed over a Cloud platform. This allows for these algorithms to be applied on real-time sensor data that is collected over the Cloud through frameworks such as the OGC SensorThings. In future, we intend to deploy this system on top of active research observatories such as the OREME's data portal.

  FIG. 2 -Proposed system architecture

  which allows for selection and crossing of numerous data streams into one data set. It sends the crossed data set to the Pattern Mining tool for extraction of gradual patterns and presents the extracted patterns to the user.

https://www.opengeospatial.org/

https://www.gostserver.xyz

https://inspire.ec.europa.eu

https://data.oreme.org

Availability of materials. The entire source code for this work is available at our GitHub repository : https://github.com/DAJOOL2020/cloud-api.git.

Summary

This paper describes a software architecture model that integrates temporal gradual pattern (GP) mining algorithms into a Cloud platform which implements OGC (Open Geospatial Consortium) SensorThings API (application interface). We build the model on top of the Sen-sorThings API in order to exploit the data streams it generates for extraction of temporal GPs.


Classification, based on energetics, of ideal icosahedral clusters, and building blocks of icosahedral quasicrystals and approximant crystals

N Tamura, J.-L.

Classification, based on energetics, of ideal icosahedral clusters, and building blocks of icosahedral quasicrystals and approximant crystals Abstract. We have classified, by their energy, compact finite clusters of icosahedral symmetry, composed of a single atom type, constituted by at most ten layers, and centered at an empty or occupied site with coordination number 12. The clusters are formed by adding one by one the best possible icosahedral layers in the surface potential minima and by relaxing numerically the cluster at each step. Polyatomic clusters are also considered. The energy is calculated from two-body pair potentials depending only upon the distance. Comparison is made between (1) the succession of layers inside these ideal icosahedral clusters, (2) the layers constituting the real clusters present in m35-approximant crystals and (3) the icosahedral local order in icosahedral quasicrystals as deduced from the 'experimental' atomic surfaces. After revisiting many m35-approximant crystals, we propose a characterization and a classification of local icosahedral order in crystallized matter and icosahedral quasicrystals.


Euan R O Allan

Benjamin Gourbal

Camila B Dores

Anaïs Portet

Christopher J Bayne

Michael S Blouin

Anais Portet

Clearance of schistosome parasites by resistant genotypes at a single genomic region in Biomphalaria glabrata snails involves cellular components of the hemolymph

Keywords: Schistosomiasis, Biomphalaria glabrata, Histology, Hemocyte, Resistance, Hemolymph transfer, Plasma transfer

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

Introduction

Schistosomiasis is a devastating and neglected tropical illness that is responsible for hundreds of thousands of deaths each year, and afflicts over 250 million people in the developing world [START_REF] Who | Schistosomiasis: population requiring preventive chemotherapy and number of people treated in 2010[END_REF][START_REF] Who | Schistosomiasis: number of people treated worldwide in 2014[END_REF]. This disease continues to persist despite mass chemotherapeutic drug administration by the World Health Organization (WHO), primarily because there are no effective vaccines targeting schistosomes, sanitation is lacking in some developing regions, and other mammalians can act as reservoir hosts and perpetuate the disease. Schistosomes require an intermediate snail host for their larval miracidial stage to develop, and regions that are unable to control these snails exhibit exacerbations in schistosomiasis cases [START_REF] Sokolow | Global assessment of schistosomiasis control over the past century shows targeting the snail intermediate host works best[END_REF]. Presently, the only areas where schistosomes have been completely eliminated are regions where these intermediate snail hosts are extirpated, which makes them an essential target for disease control [START_REF] Sokolow | Global assessment of schistosomiasis control over the past century shows targeting the snail intermediate host works best[END_REF]. In the New World, miracidia from Schistosoma mansoni infect the aquatic snail Biomphalaria glabrata, which goes on to shed cercariae capable of causing human disease. This snail species has innate immune defenses that can both specifically and nonspecifically target schistosomes for destruction [START_REF] Coustau | Advances in gastropod immunity from the study of the interaction between the snail Biomphalaria glabrata and its parasites: a review of research progress over the last decade[END_REF]. The first line of defense against miracidial penetration is the integument of the snail. The integument provides a physical barrier of connective tissue and ciliated microvillus cells that protect from invading pathogens [START_REF] Adlard | Histology and Anatomy-pathology of Molluscs: A Guide for Diagnosticians[END_REF]. Miracidia must break through this barrier before they can infect any given individual. Successful miracidia, and the sporocysts that consequently develop, are then exposed to potentially degenerating humoral factors and attacked by motile hemocytes in the hemolymph and other tissues [START_REF] Hanington | Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection[END_REF][START_REF] Loker | Gastropod Immunobiology[END_REF]. When schistosomes are recognised by hemocytes in resistant snails, they are encapsulated and exposed to anti-microbial effectors including reactive oxygen species which destroy the schistosome [START_REF] Bender | Respiratory burst of Biomphalaria glabrata hemocytes: Schistosoma mansoni-resistant snails produce more extracellular H2O2 than susceptible snails[END_REF][START_REF] Loker | Gastropod Immunobiology[END_REF]. Miracidia and sporocysts have numerous ways of evading destruction by humoral factors and hemocytes. Notable evasion tactics involve obscuring sporocyst detection post-integument penetration [START_REF] Peterson | Glycotope analysis in miracidia and primary sporocysts of Schistosoma mansoni: differential expression during the miracidium-to-sporocyst transformation[END_REF], and the production of oxidative scavengers to protect them from hemocyte-mediated damage if they are detected [START_REF] Mourao Mde | Role of the endogenous antioxidant system in the protection of Schistosoma mansoni primary sporocysts against exogenous oxidative stress[END_REF][START_REF] Mone | An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions[END_REF]. Exposed snails that are able to recognise and efficiently degrade invading schistosomes can escape infection, and subsequently avoid transmitting the disease to humans [START_REF] Portela | Evidence for specific genotype-dependent immune priming in the lophotrochozoan Biomphalaria glabrata snail[END_REF][START_REF] Pinaud | A shift from cellular to humoral responses contributes to innate immune memory in the vector snail Biomphalaria glabrata[END_REF]. Resistance of Guadeloupean B. glabrata (BgGUA) to Guadeloupean S. mansoni (SmGUA) has been shown to be strongly correlated with allelic variation in the Guadeloupe resistance complex (GRC), but not with mRNA expression levels of these genes (Tennessen et al., 2015b). The GRC is a gene region which contains novel genes with vast amino acid sequence differences and putative immune recognition functions. Grctm6 is a novel GRCencoded protein that has been shown to affect the number of cercariae shed per infected snail and is expressed in hemocytes (Allan et al., 2017b). Understanding the kinetics and biology of this new class of genes in schistosome defense is essential before they might be used in alternative schistosome control. Although these studies have highlighted the importance of the GRC during schistosome infection, little is known about the actual mechanisms by which snails bearing resistant genotypes differ from those bearing susceptible genotypes. Determining when resistant genotypes clear parasitic larvae, and whether cellular or humoral factors are involved in clearance, is essential to understanding the mechanistic role of the genes in the GRC. Histological differences between highly resistant and highly susceptible strains of snails have been characterised extensively, but these studies did not describe the genes that may cause the differences between those strains [START_REF] Richards | Transient non-susceptibility to Schistosoma mansoni associated with atrial amoebocytic accumulations in the snail host Biomphalaria glabrata[END_REF][START_REF] Borges | Histopathologic features associated with susceptibility and resistance of Biomphalaria snails to infection with Schistosoma mansoni[END_REF][START_REF] Loker | Gastropod Immunobiology[END_REF][START_REF] Nacif-Pimenta | Schistosoma mansoni in susceptible and resistant snail strains Biomphalaria tenagophila: in vivo tissue response and in vitro hemocyte interactions[END_REF]. Similarly, both cellular and humoral factors have been shown to transfer resistance from highly resistant to highly susceptible strains, but again no study has linked this transfer to allelic variation in specific genes [START_REF] Sullivan | Killing of Schistosoma mansoni sporocysts in Biomphalaria glabrata implanted with amoebocyte-producing organ allografts from resistant snails[END_REF][START_REF] Sullivan | Mitotic responses to extracts of miracidia and cercariae of Schistosoma mansoni in the amebocyte-producing organ of the snail intermediate host Biomphalaria glabrata[END_REF]Vasquez and Sullivan, 2001a,b,c;[START_REF] Pinaud | A shift from cellular to humoral responses contributes to innate immune memory in the vector snail Biomphalaria glabrata[END_REF]. In the present study, we report that snails bearing resistant GRC haplotypes have fewer unencapsulated sporocysts than snails bearing susceptible haplotypes as early as 24 h p.i., despite having equivalent integument structure and susceptibility to penetration by miracidia. We also show that resistance can be transferred from resistant GRC genotypes to susceptible GRC genotypes via injections of the resistant haplotype's hemolymph but not cell-free plasma. These findings demonstrate that GRC-mediated resistance to schistosome exposure occurs during the early stages of infection and is likely cellmediated.

Materials and methods

Maintenance, propagation, infection and inbred line derivation of B. glabrata and S.

mansoni Biomphalaria glabrata (BgGUA) and S. mansoni (SmGUA) were collected in 2005 from Dans Fond [START_REF] Theron | Effects of laboratory culture on compatibility between snails and schistosomes[END_REF] on the island of Guadeloupe, and maintained as previously described [START_REF] Theron | Multi-parasite host susceptibility and multi-host parasite infectivity: a new approach of the Biomphalaria glabrata/Schistosoma mansoni compatibility polymorphism[END_REF]Tennessen et al., 2015b). The SmGUA strain of S. mansoni was cycled through BgGUA and hamsters, and parasites were isolated from hamster livers or from shedding snails. BgGUA snails were segregated based on their genotype at the GRC locus, and separated into nine independently derived inbred lines as previously described: three RR (resistant lines R1 , R2 , R3 ), three S1S1 (susceptible lines S11 , S12 , S13 ), and three S2S2 (susceptible lines S21 , S22 , S23 ) (Tennessen et al., 2015b;Allan et al., 2017b). To produce these lines, independent founder snails, homozygous at the GRC locus, were allowed to self-fertilise for three generations. For all additional experiments, equal numbers of snails pooled from each of the three lines within a GRC genotype were used (RR, S1S1, S2S2 or RR, SS; where SS is pooled S1S1 and S2S2). Experiments were performed on size/age matched ( 7 mm, 7 weeks old) cohorts of snails which were housed identically. This research adheres to Public Health Service Domestic Assurance for humane care and use of laboratory animals (PHS Animal Welfare Assurance Number A3229-01), as Animal Care and Use Proposal 4360, and was approved by the Oregon State University Institutional Animal Care and Use Committee, USA.

Confirmation of infection phenotypes in BgGUA lines

In order to verify that the inbred lines of BgGUA that were to be used for functional work behaved phenotypically like outbred snails of the same genotype, we examined the infection phenotype of all inbred lines using the same exposure protocol that was used on outbred BgGUA (Tennessen et al., 2015b). Parasite exposures of BgGUA with SmGUA were carried out as previously described (Tennessen et al., 2015b;Allan et al., 2017b). Snails were incubated in 2 ml of dechlorinated water in individual wells of a 24 well plate containing 20 miracidia for 24 h, and then transferred into tanks containing 10-15 snails each to be monitored for infection. Three independent lines each of RR (R1 , R2 , R3 ), S1S1 (S11 , S12 , S13 ), and S2S2 (S21 , S22 , S23 ) snails were used to verify the susceptibility of these lines (Fig. 1). Infections were done on a minimum of two separate occasions with the total number of snails that were exposed exceeding 27 for any given line or treatment (n = 32 S11 ,32 S12 ,28 S13 ,35 S21 ,33 S22 ,27 S23 ,55 R1 ,31 R2 ,42 R3 ). Exposed snails were scored for cercarial shedding by placing individual snails in 2 ml of dechlorinated water in a 24 well plate for 3 h, and scored as either infected or uninfected. This was done once per week between weeks 5-10 post exposure as previously described [START_REF] Hanington | Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection[END_REF][START_REF] Hanington | A somatically diversified defense factor, FREP3, is a determinant of snail resistance to schistosome infection[END_REF]Pila et al., 2016b;Allan et al., 2017b).

Histological analysis

We histologically examined exposed snails of each genotype to determine the extent of early sporocyst development at different time points. Additionally, we examined the histological structure of the integument to ascertain if any difference in this outer barrier could explain differences in the infection phenotype of the GRC genotypes. For histological analysis of infected snails, BgGUA that were exposed to 20 miracidia were collected after 24 h, 48 h, or 10 days, and removed from their shells and fixed as previously described, with some modifications [START_REF] Pinaud | A shift from cellular to humoral responses contributes to innate immune memory in the vector snail Biomphalaria glabrata[END_REF]. In brief, two each of R1 , R2 , S11 , S12 , S21 , S22 BgGUA (n = 4 RR and 8 SS) were dissected out of their shells and fixed in Bouin's solution (75% saturated aqueous picric acid, 20% formaldehyde, 5% acetic acid; pH 1.2-1.6). After a 48 h fixation, the snails underwent consecutive 12 h washes in 95% ethanol for 36 h, and were dehydrated using consecutive 12 h washes in butanol for 36 h prior to sectioning. Fixed whole BgGUA were embedded in paraffin, underwent transverse (3 mm thick) sectioning (RHEM platform, Montpellier, France), and were stained with azocarmine G and Heidenhain's azan [START_REF] Pinaud | A shift from cellular to humoral responses contributes to innate immune memory in the vector snail Biomphalaria glabrata[END_REF]. Approximately 300 sections were stained and analysed per snail, and all sporocysts in these sections were counted. Sections underwent the following: (i) re-hydration (toluene 95, 70, 30% ethanol and distilled water); (ii) coloration (azocarmine G, 70% ethanol/1% aniline, 1% acetic alcohol, distilled water, 5% phosphotungstic acid, distilled water, Heidenhain's azan) and (iii) dehydration (95% ethanol, absolute ethanol, toluene). The preparations were then mounted with Entellan (Sigma Life Science, St. Louis Missouri, USA) and examined. All embedding, staining and histological analysis of exposed snails were completed by the group of Dr.

Gourbal at the University of Perpignan, France. For analysis of the integument of BgGUA, snails were removed from their shells and fixed in Bouin's solution as described above. Fixed snails were placed in histology cassettes and sections underwent routine diagnostic H&E staining as previouly described [START_REF] Allan | NADPH oxidase modifies patterns of MHC class IIrestricted epitopic repertoires through redox control of antigen processing[END_REF](Allan et al., , 2017a)). Slides were evaluated microscopically using a Nikon Eclipse 50i, and measurements were performed using the Nikon NIS-Elements Microscope Imaging Software. Integument thickness was measured on three distinct regions of the pedal sole from each snail. Four snails of each RR, S1S1, and S2S2 were evaluated by a veterinary pathologist (Dr. Dores) at the Veterinary Diagnostic Laboratory at Oregon State University.

Enumeration of miracidia during exposure

We wanted to know whether miracidia penetrate the three genotypes with equal frequency. Therefore, we quantified the minimum number of miracidia that did not penetrate during exposure by counting the number of miracidia remaining in an exposure well after snails were removed following 2 h exposures [START_REF] Theron | Schistosoma mansoni: distribution patterns of miracidia among Biomphalaria glabrata snail as related to host susceptibility and sporocyst regulatory processes[END_REF].

Miracidia were enumerated under a dissecting scope (n = 12 RR, 12 S1S1, 12 S2S2).

Hemolymph and cell-free hemolymph (plasma) transfer

To examine if resistance could be transferred via the direct addition of cells or endogenous molecules, S2S2 or RR hemolymph was transferred to S1S1 snails (thus avoiding injection of any genotype's own hemolymph into itself) 24 h before exposure to 20 miracidia. Two independent experimental exposures were conducted. Exposures were performed as described above with the following modifications. Hemolymph was collected from R1 , R2 , R3 S21 , S22 and S23 BgGUA snails ( 10 snails per line) and pooled by genotype prior to injection (providing pooled RR hemolymph batches consisting of hemolymph from R1 , R, and R3 ; and pooled S2S2 batches of hemolymph consisting of hemolymph from S21 , S22 and S23 ). The number of cells in the hemolymph of the three genotypes of BgGUA was determined using a haemocytometer and did not differ between groups. A small volume of sterile snail saline (SSS: 5 mM HEPES, 3.7 mM NaOH, 36 mM NaC1, 2 mM KC1, 2 mM MgCl2, 4 mM CaC12, pH 7.8) was added to ensure equal cell numbers if any discrepancy was observed on a given experimental day. Pooled RR or S2S2 hemolymph was immediately injected into S1S1 (S11 , S12 , S13 ) snails ( 15 snails/line, 20 ml/snail). Concurrently, S1S1 (S11 , S12 , S13 ) snails ( 15 snails/line, 20 ml/snail) were also injected with filter sterilised SSS as sham controls [START_REF] Adema | NADPHoxidase activity: the probable source of reactive oxygen intermediate generation in hemocytes of the gastropod Lymnaea stagnalis[END_REF]Pila et al., 2016a). As a third treatment, RR hemolymph was also left for 15 min and centrifuged for 15 min at 2000g (for cell-free plasma collection) and injected into S1S1 (S11 , S12 , S13 ) snails ( 15 snails/-line, 20 ml/snail on one occasion) (Pila et al., 2016b;[START_REF] Pinaud | A shift from cellular to humoral responses contributes to innate immune memory in the vector snail Biomphalaria glabrata[END_REF]Allan et al., 2017b). Snails that survived past week 2 for cercarial scoring exceeded 31 per group and were scored on week 5 (n = 69 S1S1 with SSS sham, n = 67 S1S1 with S2S2 hemolymph, n = 31 S1S1 with RR plasma, n = 74 S1S1 with RR hemolymph). Hemocyte only preparations were attempted, but cell viability and morphology were unacceptable for transfer after isolation. These observations are consistent with previous findings that isolation and resuspension of hemocytes can negatively affect their viability (Dr. Christopher Bayne, personal observation).

Statistical analysis

Statistical analyses were carried using GraphPad Prism software (La Jolla, CA, USA) [START_REF] Allan | Redundancy between cysteine cathepsins in murine experimental autoimmune encephalomyelitis[END_REF]. A one-way ANOVA (or unpaired Student's t-test) with a Tukey post-test was used for comparing means (P < 0.05). Before reanalysis, a natural log transformation (ln) was performed if data did not have equal variances (Barlett's or Ftest) [START_REF] Allan | Direct effects of triiodothyronine on production of anterior pituitary hormones and gonadal steroids in goldfish[END_REF]. Proportions were compared by calculating the Z score (standard score) of the population (Allan et al., 2017b).

Results

Resistance phenotypes are conserved among inbred lines of BgGUA

In the outbred BgGUA population the R allele confers an eightfold decrease in the odds of infection (Tennessen et al., 2015b). Here we verified that inbred lines derived from that outbred population behaved similarly. All three lines homozygous for the resistant allele (RR) became infected 20% of the time post-exposure, while all six families homozygous for one of the two susceptible GRC alleles (S1S1, S2S2) were infected at a rate of 80% (Fig. 1) (Tennessen et al., 2015b;Allan et al., 2017b). These lines were then used for histological and mechanistic interrogation of these genotypes.

Despite equivalent integument anatomy and miracidial penetration, resistant BgGUA have fewer free sporocysts after exposure

The integument is one of the first lines of defense during pathogenic exposure. Therefore, we considered the possibility that GRCencoded products could influence integument structure such that SmGUA larvae were less able to penetrate the integument of RR lines than SS lines. For this reason we compared, at the histological level, the integuments of SS and RR snails. Additionally, by counting miracidia left behind when snails were removed after 2 h of exposure, we collected independent data indicative of miracidial penetration of snail skin. At a histological level, the structure, cellular composition and thickness (all determined by a veterinary pathologist) of the integument did not vary among GRC genotypes (Fig. 2A,B).

Additionally, the number of miracidia unable to gain access (<1 on average) to the snail after 2h did not differ between resistant and susceptible snails, suggesting that equal numbers of miracidia penetrate these snails during an exposure (Fig. 2C). Therefore, we have no reason to believe that miracidia differentially penetrate the three genotypes.

We histologically examined snails 24 h, 48 h, and 10 days postexposure. We found no sporocysts in RR snails 10 days after exposure, so accordingly no additional analysis was conducted. After as little as 24 h, sporocysts in RR snails were clearly in the late stages of destruction, while the majority of sporocysts in SS snails were not evidently under immune attack (Fig. 3A). Indeed, the number of 'free' (not degenerating, encapsulated or under any identifiable suppression by the immune system) sporocysts was four-fold higher in SS snails (Fig. 3C). However, the total number of sporocysts identified at 24 and 48 h did not differ between RR and SS snails (Fig. 3B), an observation that is consistent with the conclusion that miracidia penetrate all three genotypes equally well (Figs. 2 and3).

Schistosome resistance is conferred to SS snails after transfer of RR hemolymph

The integument is anatomically similar in RR and SS snails and equal numbers of parasites gain access to each genotype; yet RR snails rapidly clear schistosomes whereas SS snails generally fail to do so. Therefore, we sought to determine whether the protective factor is carried in the hemolymph and, if so, whether it is humoral or cellular. We therefore attempted to transfer resistance to SS snails via RR hemolymph as it likely contains mobile hemocytes and humoral factors that may recognise and coordinate the initial innate internal defense response against schistosomes. RR hemolymph conferred a statistically significant 10% increase in resistance of S1S1 snails, while RR cell-free plasma or S2S2 hemolymph had no effect on susceptibility (Fig. 4). Therefore, cells in RR hemolymph were able to significantly improve the resistance of susceptible snails. Interestingly, the susceptibility of SS snails increased to 100% during the transfer treatment, probably owing to stress, mechanical damage or the immunological consequences of preforming injections just 24 h before exposure. Attempts to transfer isolated hemocytes were unsuccessful because the majority of isolated hemocytes did not survive isolation, resuspension and passage through a transfer needle.

Discussion

Understanding the immunological interactions between B. glabrata and schistosomes is believed to be essential to the future control of schistosomiasis [START_REF] Pearce | The immunobiology of schistosomiasis[END_REF][START_REF] Mourao Mde | Role of the endogenous antioxidant system in the protection of Schistosoma mansoni primary sporocysts against exogenous oxidative stress[END_REF][START_REF] Stefanic | RNA interference in Schistosoma mansoni schistosomula: selectivity, sensitivity and operation for larger-scale screening[END_REF][START_REF] Bhardwaj | Using RNA interference in Schistosoma mansoni[END_REF][START_REF] Valentim | Genetic and molecular basis of drug resistance and species-specific drug action in schistosome parasites[END_REF][START_REF] Theron | Multi-parasite host susceptibility and multi-host parasite infectivity: a new approach of the Biomphalaria glabrata/Schistosoma mansoni compatibility polymorphism[END_REF][START_REF] Coustau | Advances in gastropod immunity from the study of the interaction between the snail Biomphalaria glabrata and its parasites: a review of research progress over the last decade[END_REF][START_REF] Guidi | Application of RNAi to genomic drug target validation in schistosomes[END_REF]Tennessen et al., 2015a,b;Pila et al., 2016b;[START_REF] Pinaud | A shift from cellular to humoral responses contributes to innate immune memory in the vector snail Biomphalaria glabrata[END_REF][START_REF] Sokolow | Global assessment of schistosomiasis control over the past century shows targeting the snail intermediate host works best[END_REF]Allan et al., 2017b). Compatible snail species evade schistosome infection via complex physical and immunological mechanisms often involving parasite recognition [START_REF] Bender | Respiratory burst of Biomphalaria glabrata hemocytes: Schistosoma mansoni-resistant snails produce more extracellular H2O2 than susceptible snails[END_REF][START_REF] Goodall | Biomphalaria glabrata cytosolic copper/zinc superoxide dismutase (SOD1) gene: association of SOD1 alleles with resistance/susceptibility to Schistosoma mansoni[END_REF][START_REF] Hanington | Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection[END_REF][START_REF] Loker | Gastropod Immunobiology[END_REF][START_REF] Moremen | Vertebrate protein glycosylation: diversity, synthesis and function[END_REF][START_REF] Theron | Multi-parasite host susceptibility and multi-host parasite infectivity: a new approach of the Biomphalaria glabrata/Schistosoma mansoni compatibility polymorphism[END_REF][START_REF] Coustau | Advances in gastropod immunity from the study of the interaction between the snail Biomphalaria glabrata and its parasites: a review of research progress over the last decade[END_REF]Tennessen et al., 2015b;[START_REF] Pinaud | A shift from cellular to humoral responses contributes to innate immune memory in the vector snail Biomphalaria glabrata[END_REF]Allan et al., 2017b). The mechanistic bases of these interactions, and their importance to the spread of schistosomiasis by snails, are still under intensive scrutiny.

The GRC region has been shown to have a strong allelic correlation with the resistance of BgGUA to SmGUA (Tennessen et al., 2015b). Proteins in the GRC have been suggested to have an immune recognition function due to their putative structure (Tennessen et al., 2015b;Allan et al., 2017b). Furthermore, at least one protein in this region has been shown to be integral to the suppression of schistosomes in these snails and is expressed in the hemolymph (Allan et al., 2017b). Determining how the GRC influences the progression of schistosome infection is an integral part of understanding how these genes may allow some snails to be highly resistant to infection. To facilitate the functional study of resistant and susceptible individuals, homozygous BgGUA lines were developed based on their genotype at the GRC. These lines maintained the resistance phenotypes that were previously observed in the outbred population, allowing for more extensive functional assessments of infection (Tennessen et al., 2015b;Allan et al., 2017b).

In the present study, we determined that individual snails with resistant GRC genotypes destroy more compatible sporocysts in the initial days following infection than susceptible individuals, despite apparently having the same initial schistosome burden. We went on to show that some of this resistance can be transferred to susceptible snails by injecting them with resistant hemolymph, and that hemocytes are probably essential for this defensive priming. Therefore, GRC proteins are likely involved in the initial response of hemocytes to schistosome infection. After the shell, the molluscan integument is the first line of defense against most pathogens and parasites. This structure consists of an epithelium and subepithelium, which predominantly consist of ciliated cuboidal cells and secretory cells, respectively [START_REF] Adlard | Histology and Anatomy-pathology of Molluscs: A Guide for Diagnosticians[END_REF]. These cells comprise a physical barrier to invaders, as well as a motile and secretary network that can damage or remove pathogens. Although the integument has never been implicated in differences among snail strains in susceptibility to schistosomes, we decided to check for variation among genotypes in this structure to rule out the possibility that differences between GRC genotypes in thickness or structure could be causing fewer miracidia to penetrate RR snails. Additionally, given that there were few developing sporocysts (~1-4 sporocysts in 300 sections/snail) found per snail, this examination also allowed us to demine if the first physical barrier the miracidia encounter differed between genotypes and could have been partially responsible for so few sporocysts. The integuments of RR and SS BgGUA were indistinguishable with regards to cell structure, cell composition and thickness at a histological level. Accordingly, the parasitic burden was equivalent across RR and SS snails, suggesting that schistosome resistance is probably due to an immune response to sporocysts after the miracidia have penetrated the snail. It is possible that more subtle differences in the integument, or in the kinetics of schistosome penetration between genotypes, could be differentiated by electron microscopy or immunohistochemistry. However, we found similar numbers of total schistosome larvae in RR and SS snails at 24 and 48 h postexposure and similar numbers of miracidia left behind after 2 h exposures. So, it is unlikely that such subtle differences in the integument influenced the overall ability of SmGUA to penetrate the integument of BgGUA. The number of parasites found in the snails and remaining in the wells does not account for all of the parasites from the exposure. It is possible that some of these parasites were attached to the shell, or caught in the mucus of the snail and lost from our analysis. Additional microscopic methods could be used in future studies to ascertain where some of these parasites end up, and the roles that the mucus and shell may play in infection.

The developmental capacity of miracidia has been shown to be proportional to the number of miracidia that penetrate the host [START_REF] Theron | Schistosoma mansoni: distribution patterns of miracidia among Biomphalaria glabrata snail as related to host susceptibility and sporocyst regulatory processes[END_REF]. In general, sporocysts that do not survive have been shown to be encapsulated within the first 48 h, and the immune response to these sporocysts, mediated by hemocytes and humoral factors, has been shown to be faster and more extensive in more resistant strains of B. glabrata [START_REF] Theron | Schistosoma mansoni: distribution patterns of miracidia among Biomphalaria glabrata snail as related to host susceptibility and sporocyst regulatory processes[END_REF][START_REF] Pereira | Participation of cell-free haemolymph of Biomphalaria tenagophila in the defence mechanism against Schistosoma mansoni sporocysts[END_REF]. Parasite larvae which are not destroyed can be found developing weeks after an exposure in individuals that become infected, but prior to cercarial shedding. Indeed, we found that RR individuals had fewer 'free' (not encapsulated by hemocytes or degenerated by humoral factors) sporocysts despite having the same initial miracidial burden after exposure. RR snails were able to clear parasite larvae such that that no histological evidence of exposure was evident after 10 days in any of the RR snails that we examined. Given that the transfer of cell-free hemolymph has been shown to confer resistance during the initial phases of infection in other strains of B. glabrata [START_REF] Pereira | Participation of cell-free haemolymph of Biomphalaria tenagophila in the defence mechanism against Schistosoma mansoni sporocysts[END_REF][START_REF] Pinaud | A shift from cellular to humoral responses contributes to innate immune memory in the vector snail Biomphalaria glabrata[END_REF], we aimed to determine if the transfer of different hemolymph treatments could improve the resistance of SS individuals. The transfer procedure itself increased the infection outcome to 100% in control SS snails, likely due to stress, mechanical damage, hemolymph dilution, induction of an unintended immune response, or a combination of these factors. Despite this, transferred S2S2 hemolymph and RR plasma (cell-free hemolymph including humoral factors) did not alter the resistance of S1S1 snails, but RR hemolymph conferred a 10% increase in resistance.

This result implies that proteins encoded by genes in the GRC region are most likely present in RR hemocytes (as RR plasma alone did not confer resistance), and are partially responsible for the resistance phenotypes we observed and the efficient clearance of schistosome larvae by RR snails, as previously hypothesised (Tennessen et al., 2015b;Allan et al., 2017b). These results also confirm that humoral factors in RR BgGUA are probably not responsible for their greater resistance to schistosomes, but we cannot eliminate the possibility that they may work synergistically with hemocytes because we were unable to transfer a pure hemocyte preparation.

In summary, we found that schistosomes are equally likely to penetrate the three BgGUA genotypes, but that RR snails clear schistosomes much more effectively and rapidly -most within 48 h. This difference between GUA region genotypes is similar to what has been observed in comparisons between highly differentiated lines of snails that differ in their susceptibility to particular strains of S. mansoni [START_REF] Richards | Transient non-susceptibility to Schistosoma mansoni associated with atrial amoebocytic accumulations in the snail host Biomphalaria glabrata[END_REF][START_REF] Theron | Schistosoma mansoni: distribution patterns of miracidia among Biomphalaria glabrata snail as related to host susceptibility and sporocyst regulatory processes[END_REF][START_REF] Borges | Histopathologic features associated with susceptibility and resistance of Biomphalaria snails to infection with Schistosoma mansoni[END_REF][START_REF] Loker | Gastropod Immunobiology[END_REF][START_REF] Nacif-Pimenta | Schistosoma mansoni in susceptible and resistant snail strains Biomphalaria tenagophila: in vivo tissue response and in vitro hemocyte interactions[END_REF]. However, we believe this is the first time such variation can be ascribed to variation in particular genes. We postulate that proteins encoded in the GRC region are acting via hemocytes in the hemolymph because hemolymph from RR snails was able to transfer some resistance to SS individuals, while cell-free plasma was not. Previous work showed that proteins coded for by the GRC regions have structures reminiscent of immune recognition proteins and are expressed in hemolymph (Tennessen et al., 2015b;Allan et al., 2017b). These previous observations and our results here are all consistent with the hypothesis that proteins encoded in the GRC region are involved in pathogen recognition by hemocytes.

FIGURE 1

Resistance phenotypes are conserved among inbred Guadeloupean Biomphalaria glabrata (BgGUA) lines. The susceptibility of nine independent homozygous BgGUA lines (3 S1S1, S2S2 and RR) after exposure to 20 Guadeloupean Schistosoma mansoni (SmGUA) miracidia (n = 32 S11 , 32 S12 , 28 S13 , 35 S21 , 33 S22 , 27 S23 , 55 R1 , 31 R2 , 42 R3 ). Data are presented as the proportion of infected snails (± the S.E. of proportions). Significant differences (P < 0.05, Z score of proportion) are denoted by asterisks (* ).

FIGURE 2

Resistant genotypes do not have modified integument anatomy or thickness, nor do they prevent Schistosoma mansoni miracidial penetration in Guadeloupean Biomphalaria glabrata (BgGUA).

  FIGURE 3Resistant Guadeloupean Biomphalaria glabrata (BgGUA) have fewer free sporocysts, and destroy schistosomes during the initial stages of infection. (A) Free and encapsulated sporocysts in BgGUA 24 h post-exposure (scale bar = 200 mm). (B) Total number and (C) number of free (unencapsulated/undegenerated) sporocysts found in individual SS and RR BgGUA 24 h and 48 h postexposure. Data presented as means (± S.D.). Significant differences (P < 0.05, student's t-test) are denoted by asterisks (* ).

Acknowledgements

We would like to thank Jacob Tennessen, Stephanie Bollmann, Leeah Whittier, Ryan Wilson and Ekaterina Peremyslova for their technical support. This work was supported by funding from the National Institutes of Health, USA (AI109134 and AI111201) obtained by MB, and the French National Agency for Research (ANR) through a project Invimory grant (ANR-13-JSV7-0009) obtained by BG.

FIGURE 4

Transfer of resistant (RR) hemolymph, but not cell-free plasma or sensitive (SS) hemolymph, confers resistance to Guadeloupean Schistosoma mansoni (SmGUA). The susceptibility of homozygous S1S1 snails after exposure to 20 miracidia and transfer treatments. Groups of snails were treated with sham (SSS), S2S2 hemolymph (HL), RR plasma, or RR hemolymph (HL) 24 h pre-exposure (n = 69 S1S1 + sham, n = 67 S1S1 + S2S2 HL, n = 31 S1S1 + RR plasma, n = 74 S1S1 + RR HL). Data are presented as the proportion of infected snails (± the S.E. of proportions), and significant differences (P < 0.05, Z score of proportion) are denoted by an asterisk (* ).