Archives of Toxicology

, Volume 92, Issue 3, pp 1161–1176 | Cite as

Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity

  • Danielle Hagstrom
  • Siqi Zhang
  • Alicia Ho
  • Eileen S. Tsai
  • Zoran Radić
  • Aryo Jahromi
  • Kelson J. Kaj
  • Yingtian He
  • Palmer Taylor
  • Eva-Maria S. CollinsEmail author
Molecular Toxicology


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.


Acetylcholinesterase Planarians Organophosphorus pesticides Behavior Heat stress 



We thank Daniel Martinez for help and advice on the transcriptome assembly. This study was funded by the Burroughs Wellcome Fund CASI award and the Sloan Foundation (to EMSC); CounterACT Program and National Institutes of Health Office of the Director; NINDS [NS058046 (PT) and U01 NS083451 (ZR)]. DH was partially funded by the NIH Cell and Molecular Genetics Training Grant (5T32GM007240-37).

Compliance with ethical standards

Ethical standards

The manuscript does not contain clinical studies or patient data.

Conflict of interest

The authors declare that they have no conflicts of interest. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Supplementary material

204_2017_2130_MOESM1_ESM.pdf (1.1 mb)
Supplementary material 1 (PDF 1130 KB)
204_2017_2130_MOESM2_ESM.avi (157.5 mb)
Supplementary material 2 (AVI 161244 KB)


  1. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. CrossRefPubMedGoogle Scholar
  2. Arpagaus M, Fedon Y, Cousin X et al (1994) cDNA sequence, gene structure, and in vitro expression of ace-1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans. J Biol Chem 269:9957–9965PubMedGoogle Scholar
  3. Asthana S, Greig NH, Hegedus L et al (1995) Clinical pharmacokinetics of physostigmine in patients with Alzheimer’s disease. Clin Pharmacol Ther 58:299–309. CrossRefPubMedGoogle Scholar
  4. Atwood D, Paisley-Jones C (2017) Pesticides industry sales and usage 2008–2012 market estimates. U.S. Environmental Protection Agency, Washington, DCGoogle Scholar
  5. Benkert P, Biasini M, Schwede T (2011) Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27:343–350. CrossRefPubMedGoogle Scholar
  6. Bentley GN, Jones AK, Agnew A (2003) Mapping and sequencing of acetylcholinesterase genes from the platyhelminth blood fluke Schistosoma. Gene 314:103–112. CrossRefPubMedGoogle Scholar
  7. Bentley GN, Jones AK, Agnew A (2005) Expression and comparative functional characterisation of recombinant acetylcholinesterase from three species of Schistosoma. Mol Biochem Parasitol 141:119–123. CrossRefPubMedGoogle Scholar
  8. Biagioni S, Tata AM, De Jaco A, Augusti-Tocco G (2000) Acetylcholine synthesis and neuron differentiation. Int J Dev Biol 44:689–697PubMedGoogle Scholar
  9. Biasini M, Bienert S, Waterhouse A et al (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42:W252–W258. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Bigbee JW, Sharma KV, Chan EL, Bögler O (2000) Evidence for the direct role of acetylcholinesterase in neurite outgrowth in primary dorsal root ganglion neurons. Brain Res 861:354–362. CrossRefPubMedGoogle Scholar
  11. Brown DDR, Pearson BJ (2015) One FISH, dFISH, three FISH: sensitive methods of whole-mount fluorescent in situ hybridization in freshwater planarians. In: Hauptmann G (ed) In situ hybridization methods. Springer Science, New York, pp 127–150Google Scholar
  12. Camp S, Zhang L, Krejci E et al (2010) Contributions of selective knockout studies to understanding cholinesterase disposition and function. Chem Biol Interact 187:72–77. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Casida JE, Quistad GB (2004) Organophosphate toxicology: safety aspects of nonacetylcholinesterase secondary targets. Chem Res Toxicol 17:983–998. CrossRefPubMedGoogle Scholar
  14. Cebrià F, Newmark PA (2005) Planarian homologs of netrin and netrin receptor are required for proper regeneration of the central nervous system and the maintenance of nervous system architecture. Development 132:3691–3703. CrossRefPubMedGoogle Scholar
  15. Cebrià F, Kudome T, Nakazawa M et al (2002a) The expression of neural-specific genes reveals the structural and molecular complexity of the planarian central nervous system. Mech Dev 116:199–204. CrossRefPubMedGoogle Scholar
  16. Cebrià F, Nakazawa M, Mineta K et al (2002b) Dissecting planarian central nervous system regeneration by the expression of neural-specific genes. Dev Growth Differ 44:135–146. CrossRefPubMedGoogle Scholar
  17. Chu H-T, Hsiao WWL, Chen J-C et al (2013) EBARDenovo: highly accurate de novo assembly of RNA-Seq with efficient chimera-detection. Bioinformatics 29:1004–1010. CrossRefPubMedGoogle Scholar
  18. Clarke PBS, Reuben M, El-Bizri H (1994) Blockade of nicotinic responses by physostigmine, tacrine and other cholinesterase inhibitors in rat striatum. Br J Pharmacol 111:695–702. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Cochet-Escartin O, Mickolajczk KJ, Collins E-MS (2015) Scrunching: a novel escape gait in planarians. Phys Biol 12:55001. CrossRefGoogle Scholar
  20. Combes D, Fedon Y, Toutant J-P, Arpagaus M (2003) Multiple ace genes encoding acetylcholinesterases of Caenorhabditis elegans have distinct tissue expression. Eur J Neurosci 18:497–512CrossRefPubMedGoogle Scholar
  21. Cowles MW, Brown DDR, Nisperos SV et al (2013) Genome-wide analysis of the bHLH gene family in planarians identifies factors required for adult neurogenesis and neuronal regeneration. Development 140:4691–4702. CrossRefPubMedGoogle Scholar
  22. Currie KW, Molinaro AM, Pearson BJ (2016) Neuronal sources of hedgehog modulate neurogenesis in the adult planarian brain. Elife. Google Scholar
  23. Dawson RM (1994) Rate constants of carbamylation and decarbamylation of acetylcholinesterase for physostigmine and carbaryl in the presence of an oxime. Neurochem Int 24:173–182. CrossRefPubMedGoogle Scholar
  24. Eleršek T, Filipic M (2011) Organophosphorus pesticides—mechanisms of their toxicity. In: Stoytcheva M (ed) Pesticides—the impacts of pesticides exposure. Intech,  Rijeka, pp 243–260Google Scholar
  25. Ellman GL, Courtney KD, Andres V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95. CrossRefPubMedGoogle Scholar
  26. EUROSTAT (2016) Agriculture, forestry and fishery statistics—2016 edition. European Union, LuxembourgGoogle Scholar
  27. Furuhashi T, Sakamoto K (2016) Central nervous system promotes thermotolerance via FoxO/DAF-16 activation through octopamine and acetylcholine signaling in Caenorhabditis elegans. Biochem Biophys Res Commun 472:114–117. CrossRefPubMedGoogle Scholar
  28. Giacobini E (2000) Cholinesterase inhibitors: from the Calabar bean to Alzheimer therapy. In: Giacobini E (ed) Cholinesterases and cholinesterase inhibitors. Martin Dunitz Ltd, London, pp 181–219Google Scholar
  29. Gnagey AL, Forte M, Rosenberry TL (1987) Isolation and characterization of acetylcholinesterase from Drosophila. J Biol Chem 262:13290–13298PubMedGoogle Scholar
  30. González-Alzaga B, Lacasaña M, Aguilar-Garduño C et al (2014) A systematic review of neurodevelopmental effects of prenatal and postnatal organophosphate pesticide exposure. Toxicol Lett 230:104–121. CrossRefPubMedGoogle Scholar
  31. Hagstrom D, Cochet-Escartin O, Zhang S et al (2015) Freshwater planarians as an alternative animal model for neurotoxicology. Toxicol Sci 147:270–285. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Hagstrom D, Cochet-Escartin O, Collins E-MS (2016) Planarian brain regeneration as a model system for developmental neurotoxicology. Regeneration 3:65–77. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Hagstrom D, Hirokawa H, Zhang L et al (2017) Planarian cholinesterase: in vitro characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity and reactivation. Arch Toxicol 91:2837–2847. CrossRefPubMedGoogle Scholar
  34. Inoue T, Yamashita T, Agata K (2014) Thermosensory signaling by TRPM is processed by brain serotonergic neurons to produce planarian thermotaxis. J Neurosci 34:15701–15714. CrossRefPubMedGoogle Scholar
  35. Ivanović SR, Dimitrijević B, Ćupić V et al (2016) Downregulation of nicotinic and muscarinic receptor function in rats after subchronic exposure to diazinon. Toxicol Rep 3:523–530. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Kalinnikova TB, Shagidullin RR, Kolsanova RR et al (2013) Acetylcholine deficiency in Caenorhabditis elegans induced by hyperthermia can be compensated by ACh-esterase inhibition or activation of GAR-3 mAChRs. Environ Nat Resour Res 3:98–113. Google Scholar
  37. Kapka-Skrzypczak L, Cyranka M, Skrzypczak M, Kruszewski M (2011) Biomonitoring and biomarkers of organophosphate pesticides exposure—state of the art. Ann Agric Environ Med 18:294–303PubMedGoogle Scholar
  38. King AM, Aaron CK (2015) Organophosphate and carbamate poisoning. Emerg Med Clin N Am 33:133–151CrossRefGoogle Scholar
  39. King RS, Newmark PA (2013) In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea. BMC Dev Biol. PubMedPubMedCentralGoogle Scholar
  40. Layer PG, Klaczinski J, Salfelder A et al (2013) Cholinesterases in development: AChE as a firewall to inhibit cell proliferation and support differentiation. Chem Biol Interact 203:269–276. CrossRefPubMedGoogle Scholar
  41. Lenfant N, Hotelier T, Velluet E et al (2013) ESTHER, the database of the α/β-hydrolase fold superfamily of proteins: tools to explore diversity of functions. Nucleic Acids Res 41:D423–D429. CrossRefPubMedGoogle Scholar
  42. Li Y, Camp S, Rachinsky TL et al (1991) Gene structure of mammalian acetylcholinesterase. Alternative exons dictate tissue-specific expression. J Biol Chem 266:23083–23090PubMedGoogle Scholar
  43. Li B, Duysen EG, Volpicelli-Daley LA et al (2003) Regulation of muscarinic acetylcholine receptor function in acetylcholinesterase knockout mice. Pharmacol Biochem Behav 74:977–986. CrossRefPubMedGoogle Scholar
  44. Liu J, Pope CN (1998) Comparative presynaptic neurochemical canges in rat striatum following exposure to chlorpyrifos or parathion. J Toxicol Environ Health Part A 53:531–544. CrossRefPubMedGoogle Scholar
  45. Malinowski PT, Cochet-Escartin O, Kaj KJ et al (2017) Mechanics dictate where and how freshwater planarians fission. Proc Natl Acad Sci USA 114:10888–10893. PubMedGoogle Scholar
  46. Martin GG (1978) A new function of rhabdites: mucus production for ciliary gliding. Zoomorphologie 91:235–248. CrossRefGoogle Scholar
  47. Muñoz-Quezada MT, Lucero BA, Barr DB et al (2013) Neurodevelopmental effects in children associated with exposure to organophosphate pesticides: a systematic review. Neurotoxicology 39:158–168. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Mutch E, Williams FM (2006) Diazinon, chlorpyrifos and parathion are metabolised by multiple cytochromes P450 in human liver. Toxicology 224:22–32. CrossRefPubMedGoogle Scholar
  49. Nishimura K, Kitamura Y, Taniguchi T, Agata K (2010) Analysis of motor function modulated by cholinergic neurons in planarian Dugesia japonica. Neuroscience 168:18–30. CrossRefPubMedGoogle Scholar
  50. Pagán OR, Rowlands AL, Urban KR (2006) Toxicity and behavioral effects of dimethylsulfoxide in planaria. Neurosci Lett 407:274–278CrossRefPubMedGoogle Scholar
  51. Pancetti F, Olmos C, Dagnino-Subiabre A et al (2007) Noncholinesterase effects induced by organophosphate pesticides and their relationship to cognitive processes: implication for the action of acylpeptide hydrolase. J Toxicol Environ Health Part B Crit Rev 10:623–630. CrossRefGoogle Scholar
  52. Paraoanu LE, Steinert G, Klaczinski J et al (2006) On functions of cholinesterases during embryonic development. J Mol Neurosci 30:201–204. CrossRefPubMedGoogle Scholar
  53. Paz A, Xie Q, Greenblatt HM et al (2009) The crystal structure of a complex of acetylcholinesterase with a bis-(–)-nor-meptazinol derivative reveals disruption of the catalytic triad. J Med Chem 52:2543–2549. CrossRefPubMedGoogle Scholar
  54. Pezzementi L, Chatonnet A (2010) Evolution of cholinesterases in the animal kingdom. Chem Biol Interact 187:27–33. CrossRefPubMedGoogle Scholar
  55. Pezzementi L, Nachon F, Chatonnet A (2011) Evolution of acetylcholinesterase and butyrylcholinesterase in the vertebrates: an atypical butyrylcholinesterase from the medaka Oryzias latipes. PLoS One 6:e17396. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Picciotto MR, Higley MJ, Mineur YS (2012) Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous system function and behavior. Neuron 76:116–129. CrossRefPubMedPubMedCentralGoogle Scholar
  57. Pope CN (1999) Organophosphorus pesticides: do they all have the same mechanism of toxicity? J Toxicol Environ Health Part B Crit Rev 2:161–181. CrossRefGoogle Scholar
  58. Pope C, Karanth S, Liu J (2005) Pharmacology and toxicology of cholinesterase inhibitors: uses and misuses of a common mechanism of action. Environ Toxicol Pharmacol 19:433–446. CrossRefPubMedGoogle Scholar
  59. Qin YF, Fang HM, Tian QN et al (2011) Transcriptome profiling and digital gene expression by deep-sequencing in normal/regenerative tissues of planarian Dugesia japonica. Genomics 97:364–371. CrossRefPubMedGoogle Scholar
  60. Ray DE, Richards PG (2001) The potential for toxic effects of chronic, low-dose exposure to organophosphates. Toxicol Lett 120:343–351. CrossRefPubMedGoogle Scholar
  61. Rink JC (2013) Stem cell systems and regeneration in planaria. Dev Genes Evol 223:67–84. CrossRefPubMedGoogle Scholar
  62. Rink JC, Gurley KA, Elliott SA, Sánchez Alvarado A (2009) Planarian Hh signaling regulates regeneration polarity and links Hh pathway evolution to cilia. Science 326(5958):1406–1410.
  63. Rouhana L, Weiss J, Forsthoefel DJ et al (2013) RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians: methodology and dynamics. Dev Dyn 242:718–730. CrossRefPubMedPubMedCentralGoogle Scholar
  64. Russom CL, LaLone CA, Villeneuve DL, Ankley GT (2014) Development of an adverse outcome pathway for acetylcholinesterase inhibition leading to acute mortality. Environ Toxicol Chem 33:2157–2169. CrossRefPubMedGoogle Scholar
  65. Sánchez-Santed F, Colomina MT, Herrero Hernández E (2016) Organophosphate pesticide exposure and neurodegeneration. Cortex 74:417–426. CrossRefPubMedGoogle Scholar
  66. Sanders M, Mathews B, Sutherland D et al (1996) Biochemical and molecular characterization of acetylcholinesterase from the hagfish Myxine glutinosa. Comp Biochem Physiol Part B Biochem Mol Biol 115:97–109. CrossRefGoogle Scholar
  67. Selkirk ME, Lazari O, Hussein AS, Matthews JB (2005) Nematode acetylcholinesterases are encoded by multiple genes and perform non-overlapping functions. Chem Biol Interact 157–158:263–268. CrossRefPubMedGoogle Scholar
  68. Shelton JF, Geraghty EM, Tancredi DJ et al (2014) Neurodevelopmental disorders and prenatal residential proximity to agricultural pesticides: the CHARGE study. Environ Health Perspect 122:1103–1109. PubMedPubMedCentralGoogle Scholar
  69. Slotkin TA, Seidler FJ (2007) Comparative developmental neurotoxicity of organophosphates in vivo: transcriptional responses of pathways for brain cell development, cell signaling, cytotoxicity and neurotransmitter systems. Brain Res Bull 72:232–274. CrossRefPubMedPubMedCentralGoogle Scholar
  70. Soreq H, Seidman S (2001) Acetylcholinesterase—new roles for an old actor. Nat Rev Neurosci 2:294–302. CrossRefPubMedGoogle Scholar
  71. Sperling LE, Klaczinski J, Schütz C et al (2012) Mouse acetylcholinesterase enhances neurite outgrowth of rat R28 cells through interaction with laminin-1. PLoS One 7:e36683. CrossRefPubMedPubMedCentralGoogle Scholar
  72. Takano T, Pulvers JN, Inoue T et al (2007) Regeneration-dependent conditional gene knockdown (Readyknock) in planarian: demonstration of requirement for Djsnap-25 expression in the brain for negative phototactic behavior. Dev Growth Differ 49:383–394. CrossRefPubMedGoogle Scholar
  73. Taylor P (2017) Anticholinesterase agents. In: Brunton L (ed) Goodman and Gilman’s the pharmacological basis of therapeutics, 13th edn. McGraw Hill, New York, pp 163–176Google Scholar
  74. Taylor P, Radić Z (1994) The cholinesterases: from genes to proteins. Annu Rev Pharmacol Toxicol 34:281–320. CrossRefPubMedGoogle Scholar
  75. Terry AVJ (2012) Functional consequences of repeated organophosphate exposure: potential non-cholinergic mechanisms. Pharmacol Ther 134:355–365. CrossRefPubMedPubMedCentralGoogle Scholar
  76. Timofeeva OA, Roegge CS, Seidler FJ et al (2008a) Persistent cognitive alterations in rats after early postnatal exposure to low doses of the organophosphate pesticide, diazinon. Neurotoxicol Teratol 30:38–45. CrossRefPubMedGoogle Scholar
  77. Timofeeva OA, Sanders D, Seemann K et al (2008b) Persistent behavioral alterations in rats neonatally exposed to low doses of the organophosphate pesticide, parathion. Brain Res Bull 77:404–411. CrossRefPubMedPubMedCentralGoogle Scholar
  78. Umesono Y, Tasaki J, Nishimura K et al (2011) Regeneration in an evolutionarily primitive brain–the planarian Dugesia japonica model. Eur J Neurosci 34:863–869. CrossRefPubMedGoogle Scholar
  79. Waterhouse AM, Procter JB, Martin DMA et al (2009) Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191. CrossRefPubMedPubMedCentralGoogle Scholar
  80. Yang D, Howard A, Bruun D et al (2008) Chlorpyrifos and chlorpyrifos-oxon inhibit axonal growth by interfering with the morphogenic activity of acetylcholinesterase. Toxicol Appl Pharmacol 228:32–41. CrossRefPubMedGoogle Scholar
  81. Yen J, Donerly S, Linney EA et al (2011) Differential acetylcholinesterase inhibition of chlorpyrifos, diazinon and parathion in larval zebrafish. Neurotoxicol Teratol 33:735–741. CrossRefPubMedPubMedCentralGoogle Scholar
  82. Zheng D-M, Xie H-Q, Wang A-T, Wu C-C (2011) The nerve system identification by histochemical localization of acetylcholinesterase in planarian Dugesia japonica. Chin J Zool 45:68–75Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Danielle Hagstrom
    • 1
  • Siqi Zhang
    • 2
  • Alicia Ho
    • 1
  • Eileen S. Tsai
    • 1
  • Zoran Radić
    • 3
  • Aryo Jahromi
    • 2
  • Kelson J. Kaj
    • 4
  • Yingtian He
    • 1
  • Palmer Taylor
    • 3
  • Eva-Maria S. Collins
    • 1
    • 4
    • 5
    Email author
  1. 1.Division of Biological SciencesUniversity of California, San DiegoLa JollaUSA
  2. 2.Jacobs School of EngineeringUniversity of California, San DiegoLa JollaUSA
  3. 3.Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical SciencesUniversity of California, San DiegoLa JollaUSA
  4. 4.Department of PhysicsUniversity of California, San DiegoLa JollaUSA
  5. 5.Biology DepartmentSwarthmore CollegeSwarthmoreUSA

Personalised recommendations