23High-throughput screening (HTS) using new approach methods is revolutionizing 24 toxicology. Asexual freshwater planarians are a promising invertebrate model for neurotoxicity 25 HTS because their diverse behaviors can be used as quantitative readouts of neuronal function. 26Currently, three planarian species are commonly used in toxicology research: Dugesia japonica, 27Schmidtea mediterranea, and Girardia tigrina. However, only D. japonica has been demonstrated 28 to be suitable for HTS. Here, we assess the two other species for HTS suitability by direct 29 comparison with D. japonica. Through quantitative assessments of morphology and multiple 30 behaviors, we assayed the effects of 4 common solvents (DMSO, ethanol, methanol, ethyl acetate) 31 and a negative control (sorbitol) on neurodevelopment. Each chemical was screened blind at 5 32 concentrations at two time points over a twelve-day period. We obtained two main results: First, 33 G. tigrina and S. mediterranea planarians showed significantly reduced movement compared to 34 D. japonica under HTS conditions, due to decreased health over time and lack of movement under 35 red lighting, respectively. This made it difficult to obtain meaningful readouts from these species. 36Second, we observed species differences in sensitivity to the solvents, suggesting that care must 37 be taken when extrapolating chemical effects across planarian species. Overall, our data show that 38 D. japonica is best suited for behavioral HTS given the limitations of the other species. 39Standardizing which planarian species is used in neurotoxicity screening will facilitate data 40 comparisons across research groups and accelerate the application of this promising invertebrate 41 system for first-tier chemical HTS, helping streamline toxicology testing. 42 43 Keywords 44 Planarian, high-throughput screening, invertebrate, developmental neurotoxicity, solvents 45 . CC-BY-NC 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a 65 We have developed the asexual freshwater planarian Dugesia japonica as a promising new 66 invertebrate model for high-throughput neurotoxicity and DNT screening (Hagstrom et al., 2016(Hagstrom et al., , 67 2015Zhang et al., 2019aZhang et al., , 2019b. We have shown that it possesses comparable sensitivity to more 68 . CC-BY-NC 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a established new approach methods and is predictive of mammalian DNT (Hagstrom et al., 2019, 69 2015Zhang et al., 2019aZhang et al., , 2019b. The key advantage of the planarian system is its sufficiently 70 complex behavioral repertoire which enables distinct behaviors to be used as a multifaceted 71 quantitative readout of neuronal function (Hagstrom et al., 2019;Zhang et al., 2019aZhang et al., , 2019b. The 72 planarian nervous system is of medium size (~10,000 neurons), possessing >95% gene homology 73 and sharing most of the same neurotransmitte...
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