A New Method to Test Molluscicides against the Philippine Schistosomiasis Snail Vectors

To expedite the discovery of novel molluscicides in the laboratory, this study aimed to evaluate the performance of a new molluscicidal assay. This assay is based on Oncomelania hupensis quadrasi snails and is called miniaturized plate test or mpt. To perform this assay, a 12-well plate, 3 snails per well, and 24-h exposure period were used. The performance of mpt was evaluated using niclosamide and Ardisia plant extract (tagpo extract) as test substances while WHO's guidelines for a conventional plate test (cpt) served as standard. One cpt and four mpt independent trials were performed for niclosamide and tagpo extract. Probit analysis of dose–response data was run in R to generate lethal concentrations (LC50 and LC90), while lethal ratio test was performed to detect significant difference between paired LC50s (or LC90s). Using niclosamide, the calculated LC50 values were 0.104, 0.127, 0.136, 0.139, and 0.140 g/m2 for cpt, mpt 1, mpt 2, mpt 3, and mpt 4, respectively, while the LC90 values were 0.266, 0.268, 0.244, 0.251, and 0.261 g/m2, using the same sequence, respectively. For tagpo extract, the LC50 values were 1.467, 1.547, 1.659, 1.797, and 1.659 g/m2, for cpt, mpt 1, mpt 2, mpt 3, and mpt 4, respectively, and the LC90s were 2.188, 2.195, 2.501, 2.358, and 2.501 g/m2, respectively. The lethal ratio test revealed that a significant difference exists between the LC50s of cpt and mpt 1 when using niclosamide with a lethal ratio and confidence limits of 0.820 (0.663, 0.977, p < 0.05) and another significant difference between LC50s of mpt 1 and mpt 3 using tagpo extract with computed lethal ratio and confidence limits of 0.861 (0.782, 0.939, p < 0.05). Taken together, the results point out that mpt generates accurate and reproducible lethal concentration values. Hence, mpt may be used as an alternative method to screen molluscicides that are active against schistosome snail vectors.


Introduction
Population control of disease vectors is an important strategy to manage schistosomiasis, a parasitic disease caused by schistosome worms and transmitted by freshwater snails [1,2]. According to a recent modeling study, mass chemotherapy combined with snail population control is an economical strategy to eradicate schistosomiasis [3]. is mathematical model is supported by a recently published study in China. A longitudinal analysis was performed using 37 years worth of research data on schistosomiasis in Sanlian and Guifan, China [4]. In that study, a combined mass administration of praziquantel and snail control strategies resulted in significant reduction of human prevalence of schistosomiasis [4]. In China, snail control strategies include molluscicide application, environmental management, and land conversion [5]. ese strategies may be difficult to implement if snail sites vary in size and terrain or when molluscicide being used is toxic [5,6]. For example, the use of synthetic molluscicides such as niclosamide poses environmental and health risks which necessitates the development of green alternatives [6]. e broad toxicity of niclosamide to many nontarget organisms such as fish and amphibians is the major concern against its use in mass application [5]. us, there are new attempts to develop novel molluscicides by optimizing the activity of known substances or tapping diverse compounds from natural sources [7][8][9][10].
A fast in vitro molluscicidal assay may accelerate the development of novel molluscicides. ere is a growing interest in phytochemicals with potent molluscicidal activity a er the successful use of Phytolacca dodecandra in the snail population control of schistosome vectors in Egypt [7,8,11]. Synthesis of new compounds and structure-activity relationship with known molluscicides are also gaining popularity [9,10,12,13]. Both research strategies will require an assay which uses a small amount of test substance while maintaining accuracy and reproducibility. Recently, it has been shown that 2 mL of test solution in a 24-well plate can be used to screen molluscicides active against Biomphalaria species [14]. is new method was compared to the conventional protocol, a method where more than 50 mL of test solution is used for each technical replicate. Both methods were shown to generate statistically similar results [14]. e authors showed that the reduced volume of test solution needed in their new assay, in turn, reduces the amount of test substance required to generate dose-response data, making it suitable for natural products research where purified samples are always limited in amount [14].
Apart from the immersion method, molluscicides may be tested using the plate test. It is used for amphibious snails such as Oncomelania hupensis quadrasi. According to the World Health Organization's published guidelines for testing molluscicides in the laboratory, a plate test uses a 150-mm Petridish, an ordinary filter paper as flooring, ten (10) snails per plate, and a four-day exposure period [15]. ese guidelines are adapted in various ways. Its modifications may include smaller Petridish sizes, shorter exposure period, and incorporation of a revival stage where snails are allowed to recover in favorable conditions. Even if the plate test method is simple, it suffers from a large amount of test substance required in testing and extended evaluation time. is means that the plate test may be impractical when used to test bioassay-guided fractions of complex mixtures and may be difficult to reproduce as an early onset of snail death may result in microbial growth and contamination of the plate. Plate contamination may be addressed by separating out dead snails or transferring those that are alive in a new plate. To add, in WHO's testing guidelines, statistical treatment of dose-response data is not explicitly described.
Robust and appropriate statistical treatment of doseresponse data is important in assay development. Statistical treatment of data is used to model the distribution of responses sought in the assay (i.e., snail mortality rates) versus a range of concentrations of test substance. For example, Probit analysis which is used in lethal concentration calculation requires that experimentally derived dose-response data are fitted to a lognormal distribution [16]. e fitting of dose-response data depends on the model chosen and the experimental design [17]. According to Robertson et al. [18], a bioassay must use at least five concentrations and about 250 test organisms. e quality of dose-response data is assessed using goodnessof-fit tests and significance testing is performed using lethal ratio test [18,19]. In this study, robust statistical treatment of data derived from a proposed novel molluscicidal assay was performed to support its accuracy and reproducibility in calculating lethal concentrations.
is study aimed to develop a miniaturized version of WHO's plate test called the miniaturized plate test (mpt) based on Oncomelania hupensis quadrasi snails. To test the hypothesis that no significant difference exists between the lethal concentrations calculated using mpt and the conventional plate test (cpt), the reference molluscicide niclosamide and a novel plant extract were used as test substances. en, to evaluate the reproducibility of mpt, multiple trials were performed. Probit analysis was used in lethal concentration calculation, while lethal ratio test was employed in hypothesis testing [16,18].
Moreover, this study used the crude plant extract from an Ardisia species which is currently being studied by the authors for its potential molluscicidal activity against Oncomelania hupensis quadrasi snails (unpublished data). is plant is a native species in the Philippines and commonly known as "tagpo". Many Ardisia species are well studied for their health-promoting activity [20]. e purpose of including tagpo extract as a test substance is to show that mpt can be used to test crude plant extracts.

Preparation of Niclosamide.
A powder formulation of niclosamide (50% by weight) was purchased from a local dealer (NiclosM®, 50WP, Leads Agricultural Products Corp.). Seven niclosamide concentrations were used in the assay ranging from 0.0617 g/m 2 to 0.2330 g/m 2 of the active ingredient using water as solvent. To prepare the stock solution of niclosamide, 0.3530 g of 50WP niclosamide was weighed in an analytical balance (Shimadzu, Japan) and dissolved in 1 liter of distilled water using a volumetric flask. e solution was spun at 1900 × g for 4 minutes in a benchtop centrifuge (Labnet, USA). e centrifugate was stored in 50-mL centrifuge tube and stored at 4-8°C until used. e binomial name of the species was not determined due to the complexity of the genus. Green-colored tagpo fruits were brought to the lab and washed in running tap water to remove dirt. e fruits were cut into half and dried at 60°C for 24 h. Dried fruit samples were then pulverized using a blender. e pulverized fruit samples were sieved and stored in 50-mL centrifuge tubes at −21°C until used. Crude tagpo extract was prepared by soaking 1.000 g of pulverized samples in 100.0 mL of distilled water. e solid-liquid mixture was stirred continuously for 1 h and was spun at 1900 × g for 4 minutes using a benchtop centrifuge (Labnet, USA) at room temperature. e centrifugate was stored in 50-mL centrifuge tubes and used in downstream molluscicidal bioassays. e concentration of tagpo extract was expressed in grams of soluble solids per plate area (g/m 2 ) and determined using gravimetric analysis of the centrifugate following the method of the American Public Health Association [21]. For tagpo extract, five concentrations (1.224 to 2.396 g/m 2 ) were used.

Acclimatization of Snails. Snails (O. h. quadrasi)
were collected in Alang-alang, Leyte (11° 11′ 19″ N, 124° 54′ 23″ E, 49 m altitude) and acclimatized to laboratory conditions in a glass aquarium lined with filter paper on inner walls. Dechlorinated water was used for acclimatization. e water inside the aquarium was maintained at a 5 mm water depth and the water was changed every 24 h. e filter paper served as food for the snails. e snails were acclimatized under 12 h : 12 h light : dark cycles for 48 h.

Conventional Plate Test (cpt).
Molluscicidal assay was performed using 85 mm Petri plates fitted with circular filter paper on the plate surface. Appropriate volume of niclosamide or tagpo extract was distributed evenly on the filter paper.
en, the Petri plates were dried in an oven at 60°C for 6 h and cooled to room temperature. Ten snails were placed in the center of the plate and the test substance was reconstituted by distributing evenly with 2 mL of distilled water. Each test concentration was paired to one negative control. ere were as many plates for the negative control as the number of concentrations used, meaning seven plates for niclosamide and five for tagpo extract. e plates were checked a er 1 h, and snails climbing on plate walls were returned back in the center of the plate by means of tweezers. A er 24 h, the snails were assessed for mortality.

Miniaturized Plate Test (mpt).
e assay was performed using 12-well plates made from polyethylene material. e wells were fitted with 21.5 mm circular discs of filter paper. Nine out of 12 wells were added with appropriate volumes of niclosamide and tagpo extract using a micropipetor. e well plates containing the test substances were dried in an oven at 60°C for 6 h and then allowed to cool to room temperature. ree snails were added per well, a total of 36 snails were used per plate. Nine wells were used for a test substance and the remaining three wells were used as negative controls. en, 130 microliters of distilled water was added to each well to reconstitute the test substances or to make the negative control. Similar to cpt, well plates were checked a er 1 h and snails adhering on the wall were returned to the center of the well using tweezers. Snail mortality was assessed a er 24 h.

Assessment of Snail Mortality.
A er 24 h of exposure to tests substances, snails in Petri plates or 12-well plates were assessed for mortality using motility criterion. In this study, snail recovery step was skipped since we were able to assess motility of snail tissue under the stereoscope. Aside from motility criterion, the degree of decomposition and its associated foul smell were also used for mortality assessment.
To determine the count of dead snails, the snails were placed separately on a clean glass slide followed by a drop of water to each snail. e snails were crushed gently with another glass slide to expose snail tissues. e exposed tissues were prodded with a needle and lack of response a er prodding was used as the mortality criterion. As validation, we always observed noticeable reaction among snails in negative controls upon stimulation with the needle tip, that is, a er crushing between two glass slides.

Statistical Analysis.
Probit analysis or the Finney method was used to determine the median and maximal lethal concentrations (LC 50 and LC 90 , respectively), Chi-squared values, lethal ratios, and their corresponding 95% confidence limits using the drc package in R [16,22,23]. A significant difference was detected in the lethal ratio between LC 50 s (or LC 90 s) when the confidence limits of the ratio did not include 1.000 [18].

Results
In this study, a miniaturized version of WHO's conventional plate test (cpt) was developed. is new method is termed as the miniaturized plate test or mpt, which was performed with a 12-well plate (21.5 mm diameter), 3 snails per well, and 24 h exposure period to generate dose-response data. One trial of cpt and four trials of mpt were performed using seven test concentrations of niclosamide and five of tagpo extract. e researchers used WHO's conventional plate test as standard.
From the initial concentration of niclosamide and tagpo extract and the volumes used in the assay, the amount of substance used was calculated. e decrease in plate area in mpt resulted in a smaller amount of substance required to perform the assay. e amount of niclosamide used in mpt (0.0035 g) was five times lower than in cpt (0.0171 g) (Table 1). Similarly, for tagpo extract, only 0.0303 g was used for mpt and 0.1459 g for cpt. Moreover, a smaller number of snails was used in mpt than in cpt: 252 snails for mpt versus 280 for cpt with niclosamide and 180 snails for mpt versus 200 snails for cpt with tagpo extract ( Table 1).
All ten sets of dose-response data are presented in Table 2. e molluscicidal activity of niclosamide and tagpo extract was dose-dependent which means that more snails were counted as dead at higher concentrations. No snail mortality was observed in negative controls for niclosamide and tagpo extracts, which implied that the snails were competent and the mortality recorded with test substances was due to their molluscicidal action. ese results also supported that our method of snail mortality assessment by snail crushing generated accurate and reproducible dose-response data. Notably, we have observed that more snails climbed out of the

Discussion
is study reports the performance of new molluscicidal assay based on O. h. quadrasi snails using a 12-well plate, 3 snails per well, and a 24 h exposure period. is assay is called miniaturized plate test or mpt which is a modification of WHO's conventional plate test (cpt). e performance of mpt was evaluated using the calculated lethal concentrations of niclosamide and tagpo extract (a plant extract). e method mpt uses a smaller amount of test substance and a smaller number of snails compared to cpt (Table 1). is implies that the new method may be used to test the molluscicidal activity of substances with very limited sources such as synthetic products. In addition, this new method may be used to increase the throughput of testing crude plant extracts.
Philippines is a biodiversity hotspot, thus bioprospecting for molluscicidal substances from endemic plants and other sources is a worthy endeavor. Novel molluscicides discovered from Philippine natural products active against O. h. quadrasi snails such as the tagpo extract can also be tested against other medically important snail species, belonging to the genera Oncomelania, Biomphalaria, Pomacea, Lymnaea, and Bulinus.
petriplate or well plates in niclosamide especially at lower test concentrations (i.e., 0.1235, 0.0926, and 0.0617 g/m 2 niclosamide). Relative to niclosamide, there were less number of snails that climbed out with tagpo extract.
Lethal concentrations are used to compare activities of different test substances in bioassays. ese values are calculated using the Finney method (Probit analysis) or any other appropriate statistical tool [16]. e goodness-of-fit test, also known as the Chi-squared test, is a preliminary statistical treatment that validates the logistic regression model of a dose-response data [17,18]. If no significant difference is detected between the experimental data and the model, then Probit analysis becomes a valid tool to use in calculating lethal concentrations.
e results for the goodness-of-fit test are presented in Table 3. e 2 test revealed that all ten dose-response data were fitted to a lognormal distribution with all corresponding -values higher than 0.05. e calculated lethal concentrations of niclosamide and tagpo extract are presented in Table 4. e LC 50 value calculated for niclosamide was 0.104 g/m 2 with cpt and 0.127-0.140 g/m 2 with mpt. In terms of LC 90 , niclosamide had a value of 0.266 g/m 2 using cpt and 0.244-0.268 g/m 2 using mpt. For tagpo extract, an LC 50 value of 1.467 g/m 2 was calculated using cpt. e lowest LC 50 value calculated with mpt was 1.547 g/m 2 while the highest was 1.797 g/m 2 . e LC 90 value for tagpo extract was 2.188 g/m 2 and 2.195-2.501 g/m 2 determined using cpt and mpt, respectively.
Lethal ratio test was performed to determine if the difference between paired LC 50 s (or LC 90 s) is due to random errors only. If the confidence limits of a lethal ratio includes 1.000, the difference between lethal concentrations is interpreted as not significant. e lethal ratios and their corresponding confidence limits at 95% confidence level are presented in Table 5. Notably, a significant difference was detected between LC 50 s of cpt and mpt 1, 0.820 (0.663, 0.977) and LC 50 s of pair mpt 1 and mpt 3, 0.861 (0.782, 0.939). is means that changing the plate diameter from 85 mm to 21.5 mm has a significant effect on the calculated LC 50 s.  in the LC 90 s was detected with both methods either for niclosamide or tagpo extract (Table 5). Unlike in WHO's original cpt wherein bioassay data are obtained a er 4 days, mpt generates results a er 24 h only [15]. On the one hand, a 4-day exposure period may introduce uncontrolled factors in the test such as contamination of the plate from decomposing snail carcasses, on the other hand, rapid assessment of mortality will ensure that only those fast acting molluscicides will be screened and further developed. In a controlled environment (e.g., mpt) the snails are optimally exposed to the active component and an immediate onset of snail-killing effect is expected. Field conditions such as soil absorption capacity, microbial degradation, temperature fluctuation, and rainfall level may dilute the effect of an active compound so that fast-acting molluscicides may be preferred in field application. Temporal variation of molluscicidal activity may be best evaluated in field trials where the average effect of these factors are taken into account.
Moreover, the results also revealed that tagpo extract exhibited potential molluscicidal activity.
is is the first report on the molluscicidal activity of an Ardisia species. Attempts to further develop this plant extract are currently underway. Our future work would include comprehensive evaluation of the effect of different variables (e.g., exposure period and recovery step) on the accuracy and reproducibility of mpt-derived dose-response data.

Conclusions
is study revealed that the miniaturized plate test, an assay based on Oncomelania hupensis quadrasi snails, is an alternative molluscicidal assay to WHO's conventional plate test. is For example, tagpo extract is also active against the notorious rice pest, the golden apple snails or Pomacea canaliculata Lamarck, 1819 (unpublished data). Tagpo was discovered through bioprospecting of various Philippine plants for molluscicidal activity.
Similar to cpt results, the dose-response data obtained using mpt is amenable to rigorous statistical treatment which is a desirable characteristic of a bioassay. Appropriate statistical analyses of dose-response data may be used to detect systematic errors, meaning a suspected bad data due to incompetent snails or contaminated plates may be ruled out by formal mpt bioassay with niclosamide. Notably, the calculated LC 90 s for niclosamide using cpt and mpt are in good agreement with that of Wei et al. [24]. is validates the results obtained in this present study. Another interesting application of mpt is in probing of O. h. quadrasi snail resistance to widely used molluscicides. For instance, mpt can be used to evaluate snail resistance to niclosamide among different geographical isolates of snails in the country, making mpt valuable in schistosomiasis research. Taken together, these provide wider applications of this new method. e significant difference detected in the lethal ratio of paired LC 50 s as seen in Table 5 is a result of snail escaping tendency against niclosamide and the imposed reduction of plate area in mpt. is mechanism has been observed for O. hupensis snail and other species elsewhere especially at sublethal concentrations of niclosamide [24,25]. Oncomelania snails tend to escape out where niclosamide is present. is behaviour may be thought of a defense mechanism of the snails versus a potentially lethal substance. To add, even without chemical stimulus, snails can escape out of the plate faster in mpt than in cpt due to a smaller plate area. However, this does not invalidate the results since no significant difference new method offers accurate and reproducible calculation of lethal concentrations. It also uses a small amount of test substance and the dose-response data it generates can be subjected to various statistical tests (i.e., goodness-of-fit test, Probit analysis, and lethal ratio test). Further optimization of mpt is a worthy endeavor. Its future use may include the high-throughput testing of natural products (e.g., crude plant extracts) and testing potential resistance of O. h. quadrasi snails from different geographical isolates to commonly used molluscicides.

Data Availability
Data available on request. Please contact the corresponding author, Mr. Darwin Gomez. Please email at darwin.gomez@ evsu.edu.ph.

Conflicts of Interest
e authors declare that they have no conflicts of interest.