The asexual freshwater planarian Dugesia japonica has emerged as a medium-throughput alternative animal model for neurotoxicology. We have previously shown that D. japonica are sensitive to organophosphorus pesticides (OPs) and characterized the in vitro inhibition profile of planarian cholinesterase (DjChE) activity using irreversible and reversible inhibitors. We found that DjChE has intermediate features of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Here, we identify two candidate genes (Djche1 and Djche2) responsible for DjChE activity. Sequence alignment and structural homology modeling with representative vertebrate AChE and BChE sequences confirmed our structural predictions, and show that both DjChE enzymes have intermediate sized catalytic gorges and disrupted peripheral binding sites. Djche1 and Djche2 were both expressed in the planarian nervous system, as anticipated from previous activity staining, but with distinct expression profiles. To dissect how DjChE inhibition affects planarian behavior, we acutely inhibited DjChE activity by exposing animals to either an OP (diazinon) or carbamate (physostigmine) at 1 µM for 4 days. Both inhibitors delayed the reaction of planarians to heat stress. Simultaneous knockdown of both Djche genes by RNAi similarly resulted in a delayed heat stress response. Furthermore, chemical inhibition of DjChE activity increased the worms' ability to adhere to a substrate. However, increased substrate adhesion was not observed in Djche1/Djche2 (RNAi) animals or in inhibitor-treated day 11 regenerates, suggesting this phenotype may be modulated by other mechanisms besides ChE inhibition. Together, our study characterizes DjChE expression and function, providing the basis for future studies in this system to dissect alternative mechanisms of OP toxicity.

In response to noxious stimuli, planarians cease their typical ciliary gliding and exhibit an oscillatory type of locomotion called scrunching. We have previously characterized the biomechanics of scrunching and shown that it is induced by specific stimuli, such as amputation, noxious heat, and extreme pH. Because these specific inducers are known to activate Transient Receptor Potential (TRP) channels in other systems, we hypothesized that TRP channels control scrunching. We found that chemicals known to activate TRPA1 (allyl isothiocyanate (AITC) and hydrogen peroxide) and TRPV (capsaicin and anandamide) in other systems induce scrunching in the planarian species Dugesia japonica and, except for anandamide, in Schmidtea mediterranea. To confirm that these responses were specific to either TRPA1 or TRPV, respectively, we tried to block scrunching using selective TRPA1 or TRPV antagonists and RNA interference (RNAi) mediated knockdown. Unexpectedly, co-treatment with a mammalian TRPA1 antagonist, HC-030031, enhanced AITC-induced scrunching by decreasing the latency time, suggesting an agonistic relationship in planarians. We further confirmed that TRPA1 in both planarian species is necessary for AITC-induced scrunching using RNAi. Conversely, while co-treatment of a mammalian TRPV antagonist, SB-366791, also enhanced capsaicin-induced reactions in D. japonica, combined knockdown of two previously identified D. japonica TRPV genes (DjTRPVa and DjTRPVb) did not inhibit capsaicin-induced scrunching. RNAi of DjTRPVa/DjTRPVb attenuated scrunching induced by the endocannabinoid and TRPV agonist, anandamide. Overall, our results show that although scrunching induction can involve different initial pathways for sensing stimuli, this behavior’s signature dynamical features are independent of the inducer, implying that scrunching is a stereotypical planarian escape behavior in response to various noxious stimuli that converge on a single downstream pathway. Understanding which aspects of nociception are conserved or not across different organisms can provide insight into the underlying regulatory mechanisms to better understand pain sensation.

25In response to noxious stimuli, planarians cease their typical ciliary gliding and exhibit 26 an oscillatory type of locomotion called scrunching. We have previously characterized the 27 biomechanics of scrunching and shown that it is induced by specific stimuli, such as 28 amputation, noxious heat, and extreme pH. Because these specific inducers are known to 29 activate Transient Receptor Potential (TRP) channels in other systems, we hypothesized that 30 TRP channels control scrunching. We found that chemicals known to activate TRPA1 (allyl 31 isothiocyanate (AITC) and hydrogen peroxide) and TRPV (capsaicin and anandamide) in other 32 systems induce scrunching in the planarian species Dugesia japonica and, except for 33 anandamide, in Schmidtea mediterranea. To confirm that these responses were specific to 34 either TRPA1 or TRPV, respectively, we tried to block scrunching using selective TRPA1 or 35 TRPV antagonists and RNA interference (RNAi) mediated knockdown. Unexpectedly, co-36 treatment with a mammalian TRPA1 antagonist, HC-030031, enhanced AITC-induced 37 scrunching by decreasing the latency time, suggesting an agonistic relationship in planarians. 38 We further confirmed that TRPA1 in both species is necessary for AITC-induced scrunching 39 using RNAi. Conversely, while co-treatment of a mammalian TRPV antagonist, SB-366791, 40 also enhanced capsaicin-induced reactions in D. japonica, combined knockdown of two 41 previously identified D. japonica TRPV genes (DjTRPVa and DjTRPVb) did not inhibit 42 capsaicin-induced scrunching. Surprisingly, RNAi of either DjTRPAa or DjTRPVa/DjTRPVb 43 disrupted scrunching induced by the endocannabinoid and TRPV agonist, anandamide. 44 Overall, our results show that although scrunching induction can involve different initial 45 pathways for sensing stimuli, this behavior's signature dynamical features are independent of 46 the inducer, implying that scrunching is a stereotypical planarian escape behavior in response 47 to various noxious stimuli that converge on a single downstream pathway. Understanding 48 which aspects of nociception are conserved or not across different organisms can provide 49 3 insight into the underlying regulatory mechanisms to better understand pain sensation. 50 51 52Normal locomotion of freshwater planarians, termed gliding, is achieved through 53 synchronous beating of cilia in a layer of secreted mucus (1-3). Gliding planarians display a 54 smooth motion without major body shape changes, except for turning movements of the 55 anterior end. However, when exposed to certain noxious stimuli (e.g. low pH, high temperature, 56 or amputation), planarians switch to a muscular-based escape gait that is characterized by 57 oscillatory body length changes (4). We termed this gait scrunching and showed that it has a 58 characteristic set of 4 quantifiable parameters: 1. frequency of body length oscillations, 2. 59 relative speed, 3. maximum amplitude, and 4. asymmetry of body elongation and contraction 60 (4). Moreover, scrunching is conserve...