One of the key components of our proposed implementation involves Biomphalaria Glabrata being attracted to the W303 yeast in order to deliver sanguinarine. Should the molluscs not even recognize the presence of our modified yeast microorganisms, the proposed implementation would fail. Thankfully, it is a well known trick in agriculture communities that yeast can be used to attract snails, and numerous studies confirm this innate attraction (Chan et al 14). However, a precedent has not been set for whether snails actually prefer yeast over other available food options in the environment. Should Biomphalaria Glabrata ignore our yeast plates in favor of more attractive options, the proposed implementation would fail again.

As such, to demonstrate the feasibility of implementing SchistoGONE using eco-plates in the environment, we conducted tests on whether snails have a preference for yeast over other food sources. We conducted this experiment by presenting Pomacea Bridgesii snails with two options of lettuce: regular and W303 yeast-covered lettuce. By measuring the number of times the snails consume the yeast-covered lettuce, we were able to determine that the snails have an inherent preference for the presence of yeast. In the 20 trials of testing, 16 resulted in the snail selecting the yeast option. In a control test, we presented snails with two equal options of regular lettuce and randomly assigned one of the pieces to be the fake "yeast" option. The snails chose the "yeast" option by random chance, 11 out of 20 times.



In order to verify the significance of the snail preference for yeast, we ran 5000 simulations of our null hypothesis (that yeast has no attraction for snails), each consisting of 20 random decisions between yeast and non-yeast.



The sampling distribution for this proportion was then modeled using a normal curve.



By taking the expected proportion (p) as 0.5 from the 20 trials (n), the mean and standard deviation are 0.5 and 0.1118 respectively.



A z-score is then calculated, revealing our observed 0.8 proportion of yeast is in the 99th percentile should it be by random chance. Assuming the yeast we used in our experiment did not attract the snails in any capacity, there is a less than 1% chance of the observed preference for yeast lettuce occuring. The results are significant enough to reject the null hypothesis with a confidence level of 99%. As such, it can be concluded that there is a preference for W303 yeast for molluscs in our experiments.

Another key component of our proposed implementation involves our modified sanguinarine-producing microorganisms surviving environmental conditions in target geographies. Previous literature has confirmed that E.coli and W303 yeast are capable of surviving in open environments thriving in natural bodies of water (Chekabab et al 13). However, in the event of rising tides or waves crashing into the plate, it is unknown if the modified W303 yeast will be able to survive specific conditions in target locations such as Africa. Should the W303 yeast perish after short exposure to the natural conditions of the environment, the proposed implementation would fail.

As such, to demonstrate the feasibility of implementing SchistoGONE in target environments—primarily Africa—we designed a microcosm for testing the survival of yeast in conditions mimicking Lake Malawi, a notoriously schistosome infested water. We were able to replicate the pH, sedimentation, salinity, temperature, general hardness, and carbonate hardness following specifications from numerous sources of literature. Microcosms have long been used as artificial, simplified ecosystems and many studies have used this concept to test their own hypotheses on. Many of the alterations that were implemented were based off of a prior study where researchers mimicked rice paddy fields in Africa due to the locational similarities between their study and ours (Akpodiete et al 21). Our pH, sedimentation, salinity, temperature, general hardness, and carbonate hardness value replications were based on prior recorded data on Lake Malawi's characteristics in order to produce more accurate results (Massinga et al 22).

Water was sampled before, immediately after, 24 hours after, and then 48 hours after insertion of W303 s. cerevisiae, and then placed in a yeast agar plate to observe potential growth due to surviving s. Cerevisiae.



There was no presence of yeast in the initial sampling. Immediately, 24 hours, and 48 hours after yeast was inserted into the water, the samples returned notable W303 colony growth. This confirmed that yeast had survived in the simulated conditions. Because the W303 yeast was still alive in the microcosm waters, we rationalized that SchistoGONE eco-plates would remain functional in rising and turbulent tides.

One of the most important aspects of our proposed implementation is the anti-schistosomal compound killing Schistosoma sporocysts. Existing literature has confirmed sanguinarine as a cercaricidal agent, but little to no research has been done on evaluating the effects of sanguinarine and the other schistosomicides we are considering against the sporocyst stage of Schistosoma (Zhang et al 13). As such, in order to verify that the use of sanguinarine in B. glabrata snails would be able to eliminate the sporocyst phase of Schistosoma, we designed an assay to measure the survival of sporocysts over a range of given concentrations of sanguinarine, plumbagin, and continentalic acid.

Generously, Dr. Conor Caffrey at the Center for Discovery and Innovation in Parasitic Diseases (CDIPD) in the Skaggs School of Pharmacy and Pharmaceutical Sciences, UC San Diego, offered to conduct the assays for our team's project using the living Schistosoma mansoni. Dr. Caffrey designed the procedures and generated replicative data on the three anti-schistosomal compounds. From his study, he determined that sanguinarine was the most potent of the three agents tested. The proportion of living sporocysts was analyzed for different concentrations of each compound. Sanguinarine recorded a 50% effective concentration of ~0.25 µM, followed by plumbagin (EC50 ~5 µM), whereas continentalic acid was ineffective even at the highest concentration of 5 µM.

Akpodiete, N. O., & Tripet, F. (2021). Laboratory and microcosm experiments reveal contrasted adaptive responses to ammonia and water mineralisation in aquatic stages of the sibling species Anopheles gambiae (sensu stricto) and Anopheles coluzzii. Parasites & Vectors, 14(1), 1-19.

Chan, G. F., Othman, F., Zulkiffli, M. H., Yousif, R. H., Yusof, A. M., & Rashid, N. A. A. (2014). Yeasts as the Novel Attractant of Pomacea canaliculata. International Journal of Sciences: Basic and Applied Research, 18, 51-60.

Chekabab, S. M., Paquin-Veillette, J., Dozois, C. M., & Harel, J. (2013). The ecological habitat and transmission of Escherichia coli O157: H7. FEMS Microbiol Lett, 341, 1-12.

Massangi, A. V., (2022). Lake Malawi. World Lake Database : International Lake Environment Committee Foundation (ILEC),
wldb.ilec.or.jp/Display/html/3594.

Zhang, S. M., & Coultas, K. A. (2013). Identification of plumbagin and sanguinarine as effective chemotherapeutic agents for treatment of schistosomiasis. International Journal for Parasitology: Drugs and Drug Resistance, 3, 28-34.