Ruben Schols

Hans Carolus

Cyril Hammoud

Stephen Mulero

Aspire Mudavanhu e Tine Huyse
email: tine.huyse@afrikamuseum.be

A rapid diagnostic multiplex PCR approach for xenomonitoring of human and animal schistosomiasis in a 'One Health' context

Keywords: gastropod-borne disease, One Health, rapid diagnostic multiplex PCR, Schistosoma, transmission monitoring, Trematoda

Studying the epidemiology of schistosomiasis-the most prevalent gastropod-borne human disease and an economic burden for the livestock industry-relies on adequate monitoring tools. Here we describe a molecular assay for detecting human and animal African schistosome species in their planorbid gastropod host (xenomonitoring) using a two-step approach. First, schistosome infections are detected and discriminated from other trematode infections using a multiplex polymerase chain reaction (PCR) that includes a trematode-specific marker (in 18S rDNA), a Schistosoma genus-specific marker (in internal transcribed spacer 2 [ITS2]) and a general gastropod marker (in 18S rDNA) as an internal control. Upon Schistosoma sp. detection, a secondmultiplex PCR is performed to discriminate among Schistosoma haematobium, Schistosoma mansoni, Schistosoma mattheei and Schistosoma bovis/Schistosoma curassoni/Schistosoma guineensis using markers of differential lengths in the cytochrome c oxidase subunit 1 (COX1) gene. The specificity of these assays was validated with adult worms, naturally infected gastropods and human urine and stool samples. Sensitivity was tested on experimentally infected snail specimens that were sacrificed 10 and 40 days postinfection in order to mimic a natural prepatent and mature infection, respectively. The assay provides a diagnostic tool to support the xenomonitoring of planorbid gastropods for trematode infections in a One Health context, with a focus on the transmission monitoring of schistosomiasis.

Introduction

Schistosomiasis is the most prevalent gastropod-borne infectious disease in the world and causes an estimated 1.5 billion disability-adjusted life years (DALY) annually. [START_REF] Gryseels | Human schistosomiasis[END_REF] The main pathology of Schistosoma-induced infections originates from migrating eggs that cause chronic lesions in a variety of organs.1 An estimated 192 million (93%) of the worldwide estimated 207 million schistosomiasis cases occur in sub-Saharan Africa.2 Animal Schistosoma species are estimated to infect >165 million bovines in Africa and Asia, leading to substantial economic losses. [START_REF] Gower | Should we be treating animal schistosomiasis in Africa? The need for a one health economic evaluation of schistosomiasis control in people and their livestock[END_REF] Together with other gastropod-borne diseases like fascioliasis, echinostomiasis and amphistomiasis, animal schistosomiasis forms a major veterinary concern and an economic burden to the livestock industry by decreasing fertility, growth and calf survival.4,5 Schistosoma bovis, Schistosoma curassoni and Schistosoma mattheei are the most common Schistosoma species to infect livestock across sub-Saharan Africa. [START_REF] Toledo | Digenetic trematodes[END_REF] These species are also known to hybridize with Schistosoma haematobium, having important implications for schistosome epidemiology. 6-8 Besides the burden for human health and the livestock industry, trematodes can be very abundant in terrestrial and aquatic ecosystems, sometimes exceeding the total biomass of top predators,9 thereby significantly affecting ecosystem health. Moreover, several domestic bovine schistosomes also infect wild herbivores, and hybrid strains are of zoonotic importance.4,7,8 A 'One Health' approach focussing on both human and animal schistosomiasis (or trematodiases in general) is therefore desired to efficiently map trematode distribution and effectively tackle trematodiasis outbreaks in both human, domesticated and wild animal populations.3 Gastropod-borne diseases are confined by the distribution of the gastropod intermediate hosts. Insights into trematode infection rates in gastropods are thus of major epidemiological importance in trematode transmission studies. The classic method for diagnosing and quantifying trematode infections in gastropods is by inducing the natural emergence of cercariae, termed 'shedding'. However, morphological identification of larval trematode stages in gastropods is highly error-prone and difficult to impossible for genus-or species-level identification, due to the lack of diagnostic morphological traits, phenotypic plasticity and the occurrence of cryptic species.10,11 Moreover, shedding significantly underestimates the prevalence of both single and multiple species co -infections.12 This can be the result of low infection load, immature (prepatent) infections or gastropod mortality during the shedding experiment.12,13 Hence a reliable assessment of trematode infection prevalence requires a molecular diagnostic technique.12 Molecular xenomonitoring (MX) is the screening of disease vectors or intermediate hosts for the genetic material of the pathogens they transmit. [START_REF] Caron | The detection and quantification of a digenean infection in the snail host with special emphasis on Fasciola sp[END_REF] There is an ongoing debate whether polymerase chain reaction (PCR) or loopmediated isothermal amplification (LAMP) is preferred for field-sample diagnostics, as each method is characterized by specific advantages and disadvantages. 14 LAMP has proven to be a suitable method for detecting early prepatent infections in gastropod hosts.15 With specificity 10 times higher than the classic PCR, LAMP requires minimal equipment and is regarded as a fast, sensitive and cheap tool for field diagnosis.15,16 However, false negative results are observed in LAMP assays as a consequence of the method's high sensitivity to inhibition by reagents used during deoxyribonucleic acid (DNA) extraction.15 Moreover, when compared with PCR, LAMP presents less versatility for multiplexing approaches.17 Diagnostic multiplex PCR is a technique in which multiple genetic markers of differential length are amplified, allowing species-level identification up to species level. In this way, gastropod tissue samples can be screened for different trematode infections by separating the amplified DNA according to its length through agarose gel electrophoresis.18,19 Rapid diagnostic PCR (RD-PCR) refers to the use of PCR for direct diagnosis without the need for DNA sequencing, thus saving time and cutting costs.

Here we describe two PCR protocols that can be combined to identify infections by the most common African human-and livestock-infecting Schistosoma species in their planorbid gastropod host. The first multiplex PCR is an adaptation of the RD-PCR for screening Fasciola sp. infections in lymnaeid gastropods described by Carolus et al.,20 in which we added primers for Schistosoma sp. detection in planorbids. This PCR screens for general trematode infections (all trematodes) and for infections with all species belonging to the Schistosoma genus, while amplifying an internal control to avoid false negative PCR results. When positive for Schistosoma sp. infection, the second multiplex PCR can be applied to discriminate S. haematobium, S. mansoni and S. mattheei from infections of S. bovis, S. curassoni and S. guineensis. This second RD-PCR is an optimization of the assays described by Webster et

Materials and methods Samples and DNA extraction

To test and optimize the multiplex PCR assays we used experimentally infected gastropods (see 'Sensitivity testing'), naturally infected gastropods from the collection of the Royal Museum for Central Africa (RMCA, Tervuren, Belgium) and human urine and stool samples that tested positive for a S. haematobium and S. mansoni infection, respectively. The latter samples originate from a previous study in Senegal on schistosomiasis epidemiology, transmission and control (For more information see Van den Broeck et al. 21 and Meurs et al.22). The naturally infected gastropods were collected in an ongoing study in South Africa and Zimbabwe (unpublished data). In both field studies, gastropods were tested for infection by means of shedding experiments and different cercarial types that were released after light stimulus were stored in ethanol and barcoded. For snail DNA extraction, whole soft tissue was separated from the shell, ethanol was removed and the tissue was subsequently homogenized with a sterile scalpel. The total DNA of homogenates was extracted using the E.Z.N.A. Mollusc DNA Kit (OMEGA Bio-tek, Norcross, GA, USA), specially designed for DNA extraction from tissue rich in mucopolysaccharides. All DNA extracts were stored at -20°C after being diluted by a factor 10 in Milli-Q water (Merck, Darmstadt, Germany).

Primer design

The new COX1 and ITS2 primers were designed and tested in silico in Geneious 6.1.8 software (Geneious, Auckland, New Zealand) on alignments of gastropod and trematode sequences (representatives from all genera of African trematodes and for each species of Schistosoma available on GenBank Supplementary Table 1) downloaded from the National Center for Biotechnology Information (NCBI) GenBank (https://www.ncbi.nlm.nih.gov/). Primers included in the same multiplex PCR were analysed together in silico to predict the formation of potential primer dimers using the Thermo Fisher Scientific Multiple Primer Analyzer (online tool, https://www.thermofisher. com). The target region, length and sequence of each primer used are described in Table 1. Primer pairs were subsequently tested in simplex and multiplex PCR on gastropod specimens with known infection status in the two-step approach. In the first PCR, referred to as 'infection RD-PCR', three markers are amplified. Carolus et al.20 tested and described the use of the general trematode and gastropod internal control markers. This assay was adapted for Schistosoma detection via the addition of the newly designed Schistosoma sp. primer pair.

The second PCR, referred to as the 'Schistosoma RD-PCR', was designed for species discrimination of certain Schistosoma species and is only applied upon Schistosoma sp. detection in the infection RD-PCR. A universal forward primer, Asmit1,23 is combined with species-specific reverse primers (Sh.R for S. haematobium, Sman.R for S. mansoni, Smat.R for S. mattheei and Sb.R for S. bovis/S. curassoni/S. guineensis) that target markers of different lengths in the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene. This is an optimization of the protocol described in Van den Broeck et al.,21 originally based on Webster et al.,19 by adding a newly designed S. mattheei-specific primer. The species-specific reverse primers amplify bands of S. haematobium (543 bp), S. mansoni (375 bp), S. mattheei (362 bp) and S. bovis/ S. curassoni/S. guineensis (306 bp), respectively. A schematic representation of the target regions on COX1 and the respective positions of the different primers are presented in Figure 1.

PCR amplification and visualisation

All PCR reactions were performed in a 15 μL volume with 2 μL undiluted gastropod DNA extract using the Multiplex Taq DNA polymerase kit (Qiagen, Venlo, The Netherlands). Primer concentrations in the final volume were 0.2 μM 18S_Digenea_F and 18S_Digenea_R, 0.1 μM 18S_SNAIL_F and 18S_SNAIL_R and 0.6 μM ITS2_Schisto_F and ITS2_Schisto_R in the infection RDPCR. The Schistosoma RD-PCR primer concentrations in the final volume were 0.8 μM of Asmit1 universal forward primer23 and 0.3 μM of each reverse primer (Sh.R, Sb.R, Sman.R and the newly designed Smat.R primer, respectively). Both PCR protocols are performed with initial denaturation at 95°C for 3 min, 39 cycles of 94°C for 30 s, 62°C for 45 s, 72°C for 45 s and a final elongation step at 72°C for 10 min in a TProfessional Thermal Cycler (Biometra, Göttingen, Germany). Analysis of the PCR product was carried out by gel electrophoresis for 1 h 45 min at 120 V in a 3% agarose gel with Midori Green direct staining and visualization using ultraviolet light.

Validation and specificity testing

The specificity of the infection RD-PCR primers was tested on a variety of infected gastropod species from the RMCA museum collection: Biomphalaria pfeifferi infected with S. mansoni, B. pfeifferi infected with Uvulifer sp., Bulinus globosus infected with Petasiger sp., Bulinus africanus infected with S. mattheei, B. africanus with a double infection of S. mattheei and Petasiger variospinosus, Lymnaea natalensis infected with P. variospinosus and L. natalensis infected with Echinostoma sp. Infections were confirmed by sequencing COX1, ITS and 18S rDNA of the trematode infections as described in Carolus et al. [START_REF] Carolus | A cascade of biological invasions and parasite spillback in man-made Lake Kariba[END_REF] The specificity of the Schistosoma RD-PCR was tested using DNA of S. bovis, S. curassoni and S. guineensis (adult worm DNA provided by Dr Bonnie L. Webster), S. mansoni and S. haematobium (from human stool and urine samples, respectively), S. mattheei (cercariae from an infected B. africanus gastropod) and S. edwardiense (from an infected Biomphalaria sp.). The specificity of the primers Sh.R, Sb. R and Sman.R with S. intercalatum, S. margrebowiei, S. leiperi and S. rodhaini was already tested by Webster et al. [START_REF] Webster | Rapid diagnostic multiplex PCR (RD-PCR) to discriminate Schistosoma haematobium and S. bovis[END_REF] and Van den Broeck et al. [START_REF] Van Den Broeck | Optimal sample storage and extraction protocols for reliable multilocus genotyping of the human parasite Schistosoma mansoni[END_REF]. The new Smat.R primer was in silico tested for annealing with other schistosome COX1 sequences obtained from NCBI GenBank while using Geneious 6.1.8 software (Supplementary Table 1).

The following parameters were assessed to validate both PCR assays: in silico specificity of all primers, trematode species specificity of infections in different gastropod species, the capability to detect Schistosoma spp. infections in case of co-infection and the probability of false positive and false negative results. Both PCR protocols were tested at annealing temperatures of 58°C, 62°C and 65°C to validate temperature-dependent specificity and optimal annealing conditions.

Sensitivity testing

The sensitivity of both PCR protocols was tested using pure DNAextracts of Biomphalaria glabrata experimentally exposed to S. mansoni and serial dilutions thereof. The experimentally infected gastropods comprised two B. glabrata from Brazil individually exposed to a single miracidium of S. mansoni from Brazil for 24 h. These gastropods were maintained at a temperature of around 25°C, with a 12 h/12 h photoperiod and fed ad libitum, after which they were sacrificed at 10 and 40 d postmonomiracidial exposure.We prefer this method over artificially mixed host and parasite DNA, as it better reflects natural infection dynamics. The gastropods were removed from their shell and preserved in 95% ethanol. The concentration of DNA in the original DNA extract was measured using Qubit Fluorometric Quantitation (Thermo Fisher Scientific, Waltham, MA, USA). A dilution series of 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg DNA per microliter was made and PCR protocols were run.

Results

Validation and specificity

The infection RD-PCR was validated using different gastropods with and without Schistosoma sp. infections. Figure 2 shows the expected amplicons in an uninfected gastropod (Bulinus sp.) and gastropods infected with Schistosoma sp. (B. pfeifferi infected with S. mansoni) and non-Schistosoma trematodes (B. globosus infected with Petasiger sp.). DNA extracts of B. africanusinfected with S. mattheei, B. africanus with a double infection of S. mattheei and Petasiger variospinosus, L. natalensis infected with P. variospinosus, L. natalensis infected with Echinostoma sp. and B. pfeifferi infected with Uvulifer sp. all show the expected signals with the amplicon length corresponding to the internal control and general trematode markers (results not shown). The Schistosoma RD-PCR was shown to be a reliable diagnostic tool to identify infections of S. mansoni, S. haematobium and S. mattheei to the species level and to discriminate these species from S. bovis, S. curassoni and S. guineensis, as shown in Figure 3. The newly designed S. mattheei reverse primer (Smat.R) can specifically detect S. mattheei infections in their planorbid gastropod host, while it did not amplify in DNA samples from S. haematobium, S. edwardiense, S. mansoni, S. bovis, S. curassoni and S. guineensis. Due to a lack of DNA templates for the remaining Schistosoma species, we tested the specificity of the Smat.R primer in silico. These tests showed that the primer has at least 22.7% mismatching base pairs with all other schistosome species except for S. nasale (13.6%; only occurring in Asia), S. edwardiense (13.6%) and S. hippopotami (18.2%). The fact that it did not amplify in our S. edwardiense DNA sample, which showed 13.6% mismatching base pairs, suggests that it will also most likely not amplify in the remaining schistosome species, which show more mismatching base pairs. The S. bovis reverse primer (Sb.R) also amplified in the closely related species S. guineensis and S. curassoni, as already pointed out by Webster et al. [START_REF] Webster | Rapid diagnostic multiplex PCR (RD-PCR) to discriminate Schistosoma haematobium and S. bovis[END_REF]

(see below).

Both RD-PCR protocols show optimal amplification and specificity at 62°C (results not shown). We note the presence of aspecific amplification in some instances, as shown in Figure 2 (band D). We are confident that this is not the result of a co-infection, as it is also present in the experimentally infected gastropod (40-d infection) in Figure 4A. Also, its presence does not interfere with the interpretation of the infection status.

Sensitivity

The results for the sensitivity assays of the infection RD-PCR are shown in Figure 4A. The detection limit of the Schistosoma sp. marker in the experimentally infected B. glabrata was 1 ng/μL for an infection of 10 d and 10 pg/μL for a 40-day-old infection, respectively. The fact that the signal intensifies in the 40-d postexposure treatment compared with the 10-d post-exposure condition shows development and replication of S. mansoni within the snail during the course of infection. The general trematode marker showed a slightly higher sensitivity compared with the Schistosoma sp. marker and was still detectable in a total DNA dilution of 1 pg/μL and 100 fg/μL for an infection of 10 d and 40 d, respectively. The sensitivity of the S. haematobium and S. bovis primer pairs of the Schistosoma RD-PCR was rigorously tested by Webster et al. [START_REF] Webster | Rapid diagnostic multiplex PCR (RD-PCR) to discriminate Schistosoma haematobium and S. bovis[END_REF] In Figure 4B we show that the limit of detection in the Schistosoma RD-PCR is 100 pg/μL and 10 pg/μL for 10and 40-day-old S. mansoni infections, respectively.

Discussion

Molecular methods are indispensable for both trematode species discrimination10,11 and infection prevalence assessment.12 Because of their sensitivity and specificity, PCR-based techniques can be regarded as one of the most reliable, and perhaps the golden standard, of diagnostic tools for detecting specific trematode species in their intermediate host, so-called xenomonitoring. 12-14,16 Here we have developed a two-step approach to firstly detect trematode infections and then to discriminate the Schistosoma spp. involved in the gastropod infections. The 18S rDNA gene was chosen to design an internal control and a trematodespecific marker20 since it is a highly conserved region, ideal for the diagnosis of higher taxonomic levels such as the class Trematoda.10,24 The ITS2 region was targeted for Schistosoma sp. infection diagnosis because it is more variable and is capable of providing genus-specific primer annealing sites. [START_REF] Prokopowich | The correlation between rDNA copy number and genome size in eukaryotes[END_REF] The infection RD-PCR was proven to be a valid diagnostic approach regarding its specificity: Schistosoma-specific bands in gastropods with a double trematode infection including Schistosoma sp. indicate that a second trematode infection in the gastropod sample does not interfere with the detection capabilities of the infection RD-PCR method. This proves its usefulness in the diagnostic screening of field-collected gastropods, in which co-infections commonly occur.12 The combination of the Schistosomaspecific and general trematode markers in the infection RD-PCR is also a highly valuable combination to simultaneously detect Schistosoma and other economically significant trematodes such as Paramphistomoidea (a major superfamily of trematodes that infect livestock) and Diplostomoidae (a superfamily of trematodes that infect birds and fish) that use the same planorbid hosts as schistosomes.4,26

The Schistosoma RD-PCR, on the other hand, is based on COX1, a marker that is extensively used for species-level identification of animals, as this haploid-inherited mitochondrial gene evolves rapidly compared with nuclear genes in the context of speciation.27 In the Schistosoma RD-PCR, at least three species of Schistosoma occurring in sympatry can readily be detected (i.e. S. mansoni, S. haematobium and S. mattheei). This is an optimization of the protocol described by Van den Broeck et al.,21 based initially on Webster et al.,19 in which S. mattheei, one of the most important livestock schistosomes in Africa, could not be discriminated. Our new Smat.R primer appears highly specific: it did not amplify in S. haematobium, S. edwardiense, S. mansoni, S. bovis, S. curassoni or S. guineensis. Ideally, specificity tests should be conducted on all Schistosoma spp. but the fact that it did not amplify in S. edwardiense, which showed 13.6% mismatching base pairs with Smat.R, suggests that it will most likely not amplify in the remaining schistosome species, which show more mismatching base pairs in the primer annealing region. We confirmed amplification of the previously published primer Sb.R in S. guineensis and S. curassoni. This is due to their close phylogenetic relationship with S. bovis and the associated lack of sufficient COX1 sequence variability for primer design. [START_REF] Webster | Rapid diagnostic multiplex PCR (RD-PCR) to discriminate Schistosoma haematobium and S. bovis[END_REF] Increasing the annealing temperature to 65°C resolves this cross amplification but decreases the sensitivity of the protocol, as tested by Webster et al.19 S. curassoni and S. bovis infect livestock while S. guineensis infects humans in confined regions of West Africa. [START_REF] Toledo | Digenetic trematodes[END_REF] In the few geographic areas where S. bovis, S. curassoni and S. guineensis occur in sympatry, amplicon sequencing should confirm the identity of these species, as suggested by Webster et al. [START_REF] Webster | Rapid diagnostic multiplex PCR (RD-PCR) to discriminate Schistosoma haematobium and S. bovis[END_REF] We can conclude that the optimized Schistosoma RD-PCR allows identification of the most common human schistosome species present in Africa (S. mansoni and S. haematobium) from those that infect livestock (S. bovis, S. curassoni and S. mattheei) in areas where S. guineensis is not present. It can be applied on stool or urine samples or on snail samples.

The serial dilution of DNA from lab-reared B. glabrata gastropods exposed to S. mansoni showed that the PCR assays can detect parasite DNA in both mature (40-day-old) and prepatent (10-dayold) gastropod infections at DNA concentrations below the values usually obtained after DNA extraction. The general trematode marker of the infection RD-PCR appeared to be the most sensitive marker, being 100 times more sensitive (for 40-day-old infections) than the Schistosoma-specific marker used in the same assay, and the Schistosoma RD-PCR. In all the assays, the detection limit increased 10-to 100-fold in 40-day-old infections compared with 10day-old Trematodes reproduce asexually within the gastropod host, so the relative abundance of trematode DNA increases with the age of the infection. The high sensitivity of this diagnostic multiplex RD-PCR approach suggests that this assay might also be a reliable diagnostic tool for the detection of trematode DNA in human excreta (see Figure 3), as also indicated by Van den Broeck et al.,21 although standardized sensitivity and specificity tests are needed to validate this.

The infection RD-PCR described here is the first diagnostic PCR protocol for monitoring gastropod-borne trematode infections that includes a combination of an internal control, a general trematode marker and a Schistosoma-specific marker. The use of an internal control is an important asset in PCR-based diagnostics, because it excludes false negative results that commonly occur due to PCR inhibitors, failed DNA extractions, malfunctioning of thermal cyclers or an inconsistency in the preparation of the PCR mixture.28,29 Especially in studies where the sample size is large but the infection rate is low, false negative results can have a significant influence on the epidemiological conclusions drawn. [START_REF] Caron | An optimized DNA extraction and multiplex PCR for the detection of Fasciola sp. in lymnaeid snails[END_REF] We propose that an internal control should be a standard component of a diagnostic PCR protocol that is used in epidemiological studies of gastropod-borne diseases.

The absence of an internal control in the Schistosoma RDPCR is justified through the two-step approach. Samples that, for example, would gve a positive Schistosoma signal in the infection RD-PCR but fail to amplify a marker in the Schistosoma RD-PCR indicate an infection with a Schistosoma species other than the species accounted for in our Schistosoma RD-PCR, such as S. leiperi or S. margrebowiei. In that case, the identity of the infective Schistosoma species can be resolved by sequencing the amplicon of the infection RD-PCR.

The use of a general trematode marker is an interesting addition to standard PCR assays that only focus on one genus, such as Fasciola sp.20,29 or Schistosoma sp.16,19 Other taxa that potentially form a threat to humans and domesticated or wild animals are not detected in these studies and thus remain unexplored. Including a general trematode marker, although invaluable in one study (e.g. the schistosomiasis monitoring program), can be extremely valuable for other study groups that focus on other particular trematodiases (e.g. fish trematodiases).

This approach fits into the One Health framework for studying neglected tropical diseases. Since trematodiases affect the health of humans, livestock (such as cattle and farmed fish) and wild animals, they can be regarded as a burden for public, economic and environmental health. Also, the Schistosoma RDPCR fits the One Health approach as it identifies human and animal schistosomes. Both assays will therefore support the monitoring of trematode infections in different types of field samples.

Table 1.

Primers used in the multiplex RD-PCR protocols. The primer sequences and names are given along with the targeted marker, target organism and length of the amplicon (length in the number of nucleotides). The symbol '±' indicates a possible variation in amplicon length because of interspecific variation

Figure 1 .

 1 Figure 1. Schematic representation of the primers used in the Schistosoma RD-PCR and the primer location within the COX1 gene.

Figure 2 .

 2 Figure 2. Results of the infection RD-PCR assay containing different gastropod species with a variety of infections. The arrows and corresponding letters indicate the three different markers: (A) the internal control (~500 base pairs [bp]), (B) the general trematode marker (~392 bp) and (C) the Schistosoma sp. marker (~369 bp) as well as (D) aspecific amplicon. Lane 1 contains DNA of B. pfeifferi infected with S. mansoni, lane 2 contains B. globosus infected with Petasiger sp., lane 3 contains an uninfected Bulinus sp. and lane 4 contains a negative-PCR control (water). L: DNA ladder (300-3000 bp).

Figure 3 .

 3 Figure 3. The Schistosoma RD-PCR results for S. haematobium (543 base pairs [bp]) in lane 1, S. mansoni (375 bp) in lane 2, S. mattheei (362 bp) in lane 3, S. bovis (306 bp) in lane 4, S. curassoni (306 bp) in lane 5 and S. guineensis (306 bp) in lane 6. Lane 7 contains a DNA extract from a field-caught, uninfected B. africanus. S. bovis, S. guineensis and S. curassoni DNA originates from isolated adult worm specimens, the S. haematobium extract originates from an infected human urine sample, the S. mansoni extract originates from an infected human stool sample and the S. mattheei DNA originates from an infected field-caught B. africanus. Species identity was confirmed by genotyping in all samples used. L: DNA ladder (300-3000 bp).

Figure 4 .

 4 Figure 4. Sensitivity experiment for the (A) infection RD-PCR and (B) Schistosoma RD-PCR using experimentally infected B. glabrata with S. mansoni, sacrificed after 10 d (in lanes 1-9) or 40 d (in lanes 10-18) post-monomiracidial exposure. Both DNA extracts were used in undiluted form (total DNA concentration was 32.8 ng/μL 10 d post-infection and 76.7 ng/μL 40 d postinfection) and a dilution series of 10 ng (lanes 2 and 11), 1 ng (lanes 3 and 12), 100 pg (lanes 4 and 13), 10 pg (lanes 5 and 14), 1 pg (lanes 6 and 15), 100 fg (lanes 7 and 16), 10 fg (lanes 8 and 17) and 1 fg (lanes 9 and 18) per microliter. L: DNA ladder (300-3000 bp).

Acknowledgements:

We thank our colleagues from the University of Perpignan for the experimental infection of gastropod specimens, which were used in sensitivity screening. Furthermore, we would like to thank Tom Pennance and Bonnie L. Webster for their constructive comments, and thank Bonnie L. Webster and the Schistosomiasis Collection at the Natural History Museum (SCAN, London, UK) for providing the S. haematobium, S. bovis, S. curassoni and S. guineensis adult worms.

Funding:

This work was funded by the Belgian Federal Science Policy Office (BRAIN-be Pioneer Project BR/165/PI/TRAIL) and the project S1_ZMB_SNAILS_SUPPORT 2017 of the annual programme of the Cooperation Agreement dd.18.06.07 between the Belgian Development Cooperation and the Royal Museum for Central Africa. CH is funded by an FWO fellowship grant of the Research Foundation-Flanders (FWO-Vlaanderen).

Authors' contributions:

RS, HC and TH conceived the study. RS, HC, SM and TH designed the study protocol. RS, HC, CH and TH carried out the RD-PCR assays and analysed and interpreted the data. RS and HC drafted the manuscript. CH, SM, AM and TH critically revised the manuscript for intellectual content. RS and HC contributed equally to this work. All authors read and approved the final manuscript.

Competing interests:

None declared.

Ethical approval:

All experiments were performed according to the French national ethical standards established in 1 February 2013 (NOR: AGRG1238753A). Authorization was received from the French Ministere de l'Agriculture et de la Peche and the French Ministere de l'Education Nationale de la Recherche et de la technologie, permit A66040 (authorization 007083, decree 87-848).


Estelle Lebègue

Cari M Anderson

Jeffrey E Dick

Lauren J Webb

Allen J Bard

Electrochemical Detection of Single Phospholipid Vesicle Collisions at a Pt Ultramicroelectrode

We report the collision behavior of single unilamellar vesicles, composed of a bilayer lipid membrane (BLM), on a platinum (Pt) ultramicroelectrode (UME) by two electrochemical detection methods. In the first method, the blocking of a solution redox reaction, induced by the single vesicle adsorption on the Pt UME, can be observed in the amperometric i-t response as current steps during the electrochemical oxidation of ferrocyanide. In the second technique, the ferrocyanide redox probe is directly encapsulated inside vesicles and can be oxidized during the vesicle collision on the UME if the potential is poised positive enough for ferrocyanide oxidation to occur. In the amperometric i-t response for the latter experiment, a current spike is observed. Here, we report the vesicle blocking (VB) method as a relevant technique for determining the vesicle solution concentration from the collisional frequency and also for observing the vesicle adhesion on the Pt surface. In addition, vesicle reactor (VR) experiments show clear evidence that the lipid bilayer membrane does not collapse or break open at the Pt UME during the vesicle collision. Because the bilayer is too thick for electron tunneling to occur readily, an appropriate concentration of a surfactant, such as Triton X-100 (TX100), was added in the VR solution to induce loosening of the bilayer (transfection conditions), allowing the electrode to oxidize the contents of the vesicle. With this technique, the TX100 effect on

the vesicle lipid bilayer permeability can be evaluated through the current spike charge and frequency corresponding to redox vesicle collisions.

Introduction

Electrochemical detection of discrete soft nanoparticle collisions on ultramicroelectrodes (UMEs) has been recently reported as a method to determine the size distribution and the concentration of emulsion droplets, [1][2][3][4][5] vesicles, 6,7 viruses, 8 micelles 9 and biological macromolecules. 10 The observation of these stochastic events can potentially provide information on various single nanoparticles contrary to ensemble measurements. 11 Especially, electrochemical detection by collisions has been extended to studying vesicles by oxidizing the contents released upon collision at a carbon UME. 6,7 There is, however, lack of agreement about the membrane opening mechanism followed by the content electrolysis on the carbon UME.

Compton and co-workers proposed a "full collapse fusion" mechanism of the liposome membrane during the collision, based on the complete release and oxidation of ascorbic acid in a commercial vitamin C preparation, on a carbon UME. 6 In contrast, Ewing and co-workers proposed a mechanism where the vesicle first adsorbs to the electrode surface, spreads out over the electrode, and finally the oxidation of the catecholamine content occurs. 7 Moreover, this process is supported by previous investigations from Kasemo and co-workers about vesicles adsorption on various substrates (silicon dioxide, thiolated gold, oxidized gold) where the authors reported a rupture of vesicles adsorbed on hydrophobic surfaces (thiolated gold) and by opposition, an adsorption of intact vesicles on more hydrophilic surface (oxidized gold). 12,13 In the same trend, Scholz and co-workers studied the behavior of different liposomes on the electrode surface (mainly a mercury electrode) and showed that chronoamperometric measurements are an interesting tool to probe the membrane stability and to understand the effect of its properties on vesicle fusion. [14][15][16][17] Especially, the authors were the first to study the adhesion and bursting processes of 450 nm diameter liposomes, and the spreading of the lecithin on the mercury surface (0.48 mm 2 ) by using chroamperometric measurements for determining the frequency of spikes and the charge densities of formed monolayers. [14][15][16] To improve our understanding of vesicle interactions with an electrode surface, we herein present different vesicle behavior on a Pt UME, which serves as a hydrophilic surface for vesicle collisions. By analogy with emulsion results, [1][2][3][4] we report two techniques of vesicle detection: (1) vesicle blocking (VB), which consists of observing a blocking of solution redox species due to the single vesicle adsorption on the UME, and (2) vesicle reactor (VR), where the redox probe is encapsulated inside the vesicle and can be electrolyzed at the UME after its collision (Figure 1). In both cases, the redox probe chosen was potassium ferrocyanide [K4Fe(CN)6] 18 because of its high solubility in water (0.5 M). Moreover, 0.5 M K4Fe(CN)6 can be easily encapsulated inside vesicles as a hydrophilic content and removed from the solution outside vesicles. Also, it is a relevant redox species for the continuous phase largely used for detection of various soft nanoparticle collisions by the blocking method. 1,8,10 Here we demonstrate that the BLM does not break and release the contents during vesicle collision at the Pt UME hydrophilic surface. As shown in previous studies of BLMs, 19,20 they do not allow effective electron tunneling or ion transport to cause redox reactions across them without the presence of a strong surfactant. However cell transfection experiments show that the presence of a surfactant, e.g., Triton X-100 (TX100), can promote transfer across the membrane. 18,21 Moreover, we clearly show that vesicles are adsorbed on the Pt surface (VB) after their collision, but the electron transfer cannot happen through the bilayer for electrolyzing the Fe(CN)6 4-content (VR). Hence, we discuss here the TX100 concentration effect in solution and also the corresponding kinetic study on the vesicle membrane permeability by detection of electrochemical collision events. Our results were in good agreement with several studies reporting the role of TX100 surfactant on the cell membrane, which could yield fundamental insight into the transfection process. [21][22][23][24][25] To the best of our knowledge, this is the first work dealing with the vesicle collision behavior onto a hydrophilic surface.

Experimental Section

Reagents. All chemicals were reagent grade and used as purchased without further purification. Water used in each experiment was Milli-Q water. Chloroform (≥99.8%), sulfuric acid (97%) and hydrogen peroxide (30%) were obtained from Fisher Scientific.

Ferrocenemethanol (97%), potassium ferrocyanide, potassium phosphate monobasic (≥99.0%), potassium phosphate dibasic (≥98%) and Triton X-100 were purchased from Sigma Aldrich. Pt Platinum ultramicroelectrodes were prepared by laser pulling (Sutter Instruments) according to the general procedure performed in our lab, 26,27 followed by mechanical polishing with Bevellers for a diameter between 1 and 2 μm. Before each experiment, the Pt UMEs were washed by dipping in piranha solution (mixture composed to 3:1 concentrated sulfuric acid to 30% hydrogen peroxide solution) for ten seconds, and then dipping in water, and finally dipping successively in acetone, ethanol and several times in water. The radius of the Pt UME was obtained using the steady-state current in cyclic voltammetry recorded in 1 mM ferrocenemethanol aqueous solution. The experimentally observed frequency of current steps at the Pt UME with the VB method was 0.12 Hz. The vesicles concentration (Cves) can be calculated from this collision frequency of vesicles onto the UME surface by eq 1, based on a diffusion-limited flux of nanoparticles to the electrode surface. 28 The concentration of the pure DMPC vesicles aqueous solution before dilution is evaluated as 5.5 ± 0.2 nM, a value in good agreement with the concentration determined by NTA (Figure S2) at 3.1 ± 0.2 nM, suggesting that diffusion is the dominant process compared with migration effects. 29 A small contribution of mass transfer of the vesicles to the electrode surface by electrophoretic migration cannot be ruled out; however, because the concentration of potassium ferrocyanide is 0.2 M, it likely carries most of the current, making diffusion the predominate form of mass transfer. 8 Cves = 𝑓 ves 4𝐷 ves 𝑟 e 𝑁 A

Results and Discussion

where fves is the collision frequency by diffusion of the vesicles to the UME, Dves is the diffusion coefficient of a spherical vesicle, re is the radius of the working electrode, and NA is Avogadro's number. The diffusion coefficient (Dves) can be estimated by the Stokes-Einstein relation (eq 2) 30 Dves

= 𝑘 B 𝑇 6𝜋𝜂𝑟 ves ( 2
)
where kB is Boltzmann's constant, T is temperature, η is the viscosity of the continuous phase at 25 °C, and rves is the hydrodynamic radius of a vesicle. From this equation, the diffusion coefficient of a 113 nm diameter vesicle (determined by DLS measurement in Figure S1) is 4.3 × 10 -8 cm 2 s -1 , matching well with the value estimated from NTA data (Figure S2) at 4.1 × 10 -8 cm 2 s -1 .

The current steps shown in Figure 2 are related to the single vesicle collisions at the Pt UME surface. In most cases, we observed a current decrease in the shape of a stair step, indicating that most of the vesicles stick on the Pt surface after collision and only in rare cases, they quickly leave the UME after collision, showing a current increase in the shape of a stair step. Because of these observations, we assume that the probability of the adsorbate sticking to the electrode is nearly 1. Therefore, the VB electrochemical method is a relevant technique to detect nanometer-sized vesicles at a Pt UME and can give some interesting information such as the vesicles solution concentration and also information on the vesicle's adhesion on the Pt surface. The sharpness of the steps (rise time 0.25 s) suggests that any vesicle distortion or spreading either occurs very quickly, or more likely, that the vesicle maintains its essentially spherical shape. VR method. The electrochemical detection of collisions by vesicles reactor method is presented in Figure 3. The main observation in Figure 3 was the absence of current spikes in the chronoamperometric i-t curve recorded in the presence of DMPC vesicles without surfactant. Indeed, this curve showed a similar shape to the one recorded in aqueous phosphate buffer solution (in the absence of vesicles solution) with the same trend to reach the steadystate current. This result shows no oxidation of the vesicles content occurred during each collision, suggesting no lipid bilayer collapse against the Pt UME. According to the VB method applied to redox DMPC vesicles (Figure S4), we can affirm that vesicles irreversibly adsorb onto the Pt UME, but the VR technique presented in Figure 3 clearly demonstrated that electron transfer does not occur through the BLM because electron tunneling and ion transfer is not facile through such a thick barrier. 19,20,31 Thus, in agreement with previous studies about the vesicle adsorption on various substrates, 12,13 DMPC vesicles seem to remain intact during their collision on the Pt UME hydrophilic surface.

In this case, to increase the membrane permeability, the use of a surfactant was required.

The i-t curve recorded immediately after addition of an appropriate concentration of TX100 (vide infra, Figure 4) showed several current spikes over 300 seconds (Figure 3) with a frequency estimated at 0.28 Hz. Note the narrow shape of these current spikes detected at Pt UME, that are clearly different to those observed in previous studies. 6,7 Indeed, contrary to the expected "blip" shape (Figure 1), here the current spikes present a symmetrical and sharp shape for a time ranging between 0.2 to 0.5 seconds. This result indicates that mechanism occurring during single vesicle collisions at Pt UME in the presence of an appropriate concentration of TX100 is probably different to those reported by Compton and Ewing at a carbon UME. 6,7 Moreover, this short time observed for current spikes suggests a quick releasing of the content against the Pt UME, in a rapid electrolysis process. Thus, the electrolysis mechanism of the DMPC vesicles content during the collision at the Pt ultramicroelectrode surface in the presence of surfactant seems more complicated than a simple releasing process and probably requires additional studies to improve the understanding.

As previously shown in the VB technique, the vesicles concentration (Clip) can be calculated by using eq 1 and eq 2 based on 120 nm diameter vesicles (determined by DLS measurement in Figure S1 and NTA data in Figure S2). Hence, the concentration of the pure redox DMPC vesicles aqueous solution before dilution is evaluated at 4.4 ± 0.1 nM, a value close to that determined by NTA (Figure S2) at 3.3 ± 0.2 nM and also in good agreement with the vesicles concentration determined by the VB method (Figure 2). This result indicates again that migration effects are less significant than diffusion, which is expected because very little faradaic current is flowing during VR experiments. 29 Moreover, to confirm that current spikes are due to oxidation of Fe(CN)6 4-contained inside the vesicle during its collision, we can estimate the diameter of each vesicle from the charge obtained by calculating the charge passed during the collision, which corresponds to the amount of ferrocyanide oxidized and using Faraday's Law. Here, we assume that the initial concentration of ferrocyanide introduced into the vesicle solutions before extrusion is the same in each vesicle (i.e., an attoliter aliquot of the original solution), and we also assume that the electrode consumes all of the contents of the vesicle. In addition, we considered the cutoff for a "signal" (current spike) when the spike was at least three times the background noise in current. A background experiment without redox species inside DMPC vesicles (Figure S5)

showed no current spike.

Thus, the vesicle diameter (dves) can be calculated by eq 3:

dves = 2 √ 3𝑄 4𝜋𝑛 e 𝐹𝐶 redox 3 (3)
where Q is the measured charge, ne is the number of electrons transferred during the electrolysis, ne = 1, F is Faraday's constant, and Credox is the concentration of redox species encapsulated in vesicle, Credox = 0.5 M.

The overlay presented in Figure 4a showed that the size distributions from DLS data and calculated data were quite similar. Indeed, the DLS data indicated that the peak diameter is 120 ± 30 nm (Figure S1) while the calculated data gave 116 ± 63 nm as mean diameter from the corresponding average charge estimated at 0.079 pC. According to this result, the previous assumptions about the complete electrolysis and the expected concentration of ferrocyanide encapsulated inside vesicles (0.5 M) can be validated and in addition, that showed the TX100 surfactant does not act on the size distribution of vesicles (nor on the consumed charge) during their collision.

As expected, the VR technique confirmed its efficiency to determine the size distribution of nanometer-sized vesicles and was also relevant to approximate the vesicles concentration in solution. Nevertheless, this last technique provided clear evidence observation of current spikes form these vesicles on a Pt UME necessitated a surfactant, such as TX100, in a controlled concentration. Indeed, TX100 is one of the most widely used nonionic surfactants to permeate the living cell membrane for transfection. 21,24 In fact, the transfection process consists to the opening of pores or holes in the BLM to allow the hydrophilic species adsorption/desorption of the vesicle content without damaging the membrane. 24 The study of TX100 surfactant concentration effect on the redox DMPC vesicle collisions frequency by detection of corresponding current spikes on Pt UME is presented in Figure 4b, to determine the appropriate TX100 concentration to use.

Figure 4b shows the evolution of the collisions frequency versus the TX100 concentration added every 5 minutes in the redox DMPC vesicles aqueous solution by recording a chronoamperometric i-t curve (VR method) after each new addition. Especially, a chronoamperometric i-t curve like the one presented in Figure 3 is recorded on 300 seconds every 5 minutes (after each new addition of TX-100) on a fresh and clean Pt UME and the collisions frequency (number of collisions during 300 seconds) is reported in Figure 4b for each TX-100 concentration added. The collisions frequency began at 0 Hz for TX100 concentrations below 0.14 mM and increased until the appropriate TX100 concentration at 0.18 mM provided a maximum frequency value of 0.065 Hz. These concentrations values are in good agreement with the previous work of Koley and Bard reporting the TX100 concentration effects on membrane permeability of a single HeLa cell, where the TX100 critical micelle concentration (CMC) was evaluated at 0.17 mM. 21 According to their study, if the TX100 concentration is above this CMC (>0.18 mM) the cell membrane is irreversibly damaged. Indeed, above 0.18 mM in Figure 4b, the collisions frequency quickly decreased and after 0.25 mM TX100 almost no current spike was detected, suggesting the fatal breaking of the vesicles lipid bilayer.

Unfortunately, the concentration of ferrocyanide released in solution following the prospective collapsing of all vesicles is too low (~ 7 μM) for inducing a significant difference on the background steady state current in 0.1 M KPB aqueous solution. Several experiments have been performed concerning the TX100 concentration effect on single vesicle collisions and in all cases the collisions frequency curves versus the TX100 concentration (Figure S6) have presented the same trend observed in Figure 4b with a rapid increase at the appropriate TX100 concentration value around 0.20 ± 0.03 mM. Moreover, all chronoamperometric i-t curves recorded in these conditions showed the same shape and size of current spikes confirming that surfactant does not disturb the electrolyzed content (integrated charge). Here, the TX100 effect on the vesicles BLM permeability could be explained by a process that involves the more facile release of the ferrocyanide content only during vesicle collision.

Conclusion

In summary, we have investigated vesicle behavior composed of a phospholipid bilayer membrane on a Pt ultramicroelectrode surface by two electrochemical detection methods involving discrete collision events. For the first time, we have shown that the vesicles blocking method is a useful technique for observing the vesicle adhesion on the Pt UME surface and also to evaluate the vesicle concentration in solution. Furthermore, our results have confirmed that the vesicle reactor method can be used to determine the vesicle size distribution, but the addition of a surfactant like Triton X-100 is necessary to oxidize the DMPC vesicle content. Indeed, in the absence of surfactant, the BLM does not allow passage of the contents to the UME for electrolysis during the vesicle collision on the Pt UME and is also too thick for allowing electron transfer across the BLM. Therefore, we have shown that the required Triton X-100 concentration is about 0.20 ± 0.03 mM depending on the DMPC vesicle concentration in solution and under these conditions the BLM is permeable to hydrophilic species such as potassium ferrocyanide. Finally, we have suggested a transfection mechanism from the surfactant to explain the current spikes occurring during vesicles reactor collisions at Pt

Figure 1 .

 1 Figure 1. Schematic representation of the two reported vesicle collision techniques at a Pt UME

( 99 .

 99 9%) wire was obtained from Goodfellow (Devon, PA). 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) lipids were purchased in powder from Avanti Polar Lipids and stored in a freezer.Vesicle preparation. Unilamellar vesicles solutions were prepared by dissolution of 10 mM DMPC lipid (powder) in chloroform (1 mL), then vortexed for 5 minutes and placed into a warm water bath (40 °C) for at least 10 minutes until the complete dissolution of lipids. The homogeneous mixture was placed under ambient atmosphere overnight and then, under vacuum for 1 hour for the complete evaporation of chloroform. The dry lipid film was hydrated by addition of aqueous solution (2 mL of pure water or 2 mL of 0.5 M K4Fe(CN)6 aqueous solution) and then, the solution was mixed for 5 minutes and heated on a hot plate at 40 °C for 30 minutes. The DMPC vesicles solutions were extruded using 400 nm, 200 nm and finally 100 nm diameter polycarbonate membranes. The vesicles solution was passed through the extruder, which was kept warm at 40 °C, and then were passed through the extruder 10 times (for each polycarbonate membrane size) to obtain monodisperse nanometer-sized DMPC unilamellar vesicles solutions. The final step was to pass DMPC vesicles solution through a column (PD-10 Desalting Columns, GE Healthcare) by using pure water for removing K4Fe(CN)6 outside vesicles.Materials and Instrumentation.The vesicles extrusion phase was carried out with the extruder set from Avanti Polar Lipids including a mini-extruder, 2 syringes of 1 mL, polycarbonates membranes of 0.4 μm, 0.2 μm and 0.1 μm, and filter supports. The electrochemical experiments were performed using a CHI model 920C and CHI630 potentiostat (CH Instruments, Austin, TX) with a three-electrode cell placed in a faraday cage. Platinum wire was used as a counter electrode and the reference electrode was Ag/AgCl. For all chronamperometric i-t curves recorded, the sample interval (in sampling time) was 50 ms.Dynamic light scattering (DLS) data was obtained by Zetasizer Nano ZS (Malvern, Westborough, MA) and nanoparticle tracking analysis (NTA) data was achieved by Nanosight.However this last one (NTA) was discontinued when we found fouling of the cell window. The size distribution of vesicles was analyzed by DLS in FigureS1and NTA in FigureS2, both in agreement with 120 ± 30 nm diameter DMPC vesicles aqueous solutions.

Figure 2 .

 2 Figure 2. (A) The i-t curve for collision experiments by vesicles blocking method recorded at

Figure 3 .

 3 Figure 3. The i-t curve for collision experiments by vesicles reactor method recorded at +0.6

Finally, our studyFigure 4 . 1 M

 41 Figure 4. (a) Size distributions from DLS data (black line) and from charge data by integrating

Acknowledgments

EL acknowledges the Defense Threat Reduction Agency (Grant No. HDTRA1-11-1-0005) and also, Christophe Renault and Jiyeon Kim for helpful advices. JD acknowledges the National Science Foundation Graduate Research Fellowship (Grant No. DGE-1110007). AB acknowledges the National Science Foundation (Grant No. CHE-1405248) and the Welch Foundation (Grant No. F-0021). LW acknowledges the Welch Foundation (Grant No. F-1722).

ultramicroelectrode but this assumption should be checked by extending this study to other surfactants commonly used for cell transfection and probably also to other encapsulated hydrophilic electroactive species.

Supporting Information

Additional figures are available in the supplementary information.

Corresponding Author

ajbard@cm.utexas.edu

Competing Financial Interest

The authors declare no competing financial interest.