AbstractBery,A.andMarti´nez, P. 2010. Acetylcholinesterase activity in the developingand regenerating nervous system of the acoel Symsagittifera roscoffensis. —ActaZoologica (Stockholm) 00:1–10.The use of the cholinergic system is wid espread in the animal kingdom. It con-trols different processes, including reproduction and neural transmission. How-ever, its evolutionary history is not yet well understood. For instance, the roleplayed by the cholinergic system in the nervous system of basal bilaterian taxa,where the first signs of architectural complexity appear, is still unknown. Here,we describe the structure of the cholinergic system during the development andregeneration of the acoel flatworm Symsagittifera roscoffensis, using acetylcholines-terase (AchE) activity as a marker. In this species, AchE activity is observed at alldevelopmental stages, including in the early embryos. The juvenile and adultpatterns reveal the presence of a comple x nervous system that includes threepairs of longitudinal neurite bundles, whi ch are connected to an anterior central-ized mass of neurons and neural processes formed by two pairs of connectivesand four commissures. The power of the technique also allows the detection ofnewly born neurons as they are incorpo rated into the growing nervous system(during regeneration).Pedro Marti´nez, Departament de Gene`tica, Universitat de Barcelona, Av. Diag-onal, 645. E- 08028-Barcelona. Spain. E-mail: Pedro.martinez@ub.eduIntroductionAcetylcholine (Ach) is a molecule that has been detected inbacteria, fungi, plants and animals (Horiuchi et al. 2003).The synthesis of the neurotransmitter Ach is modulated bythe enzymes choline acetyl transferase and carnitine acetyltransferase. Conversely, the determination of neurotransmis-sion at cholinergic synapses is produced by the hydrolysis ofthis neurotransmitter mediated by the enzyme acetylcholines-terase (AchE) (Silman and Sussman 2008). The biologicalfunction of Ach has been studied extensively in bilaterians,especially insects and vertebrates (Le Nove`re et al. 2002).However, in organisms without a nervous system and in morebasal metazoans, the physiological function of Ach has yet tobe determined, although it may be a local mediator.Moreover, even in biological systems that have a well-orga-nized nervous system, the presence of components of the cho-linergic system is not restricted to neurons (Ellwanger andNickel 2006; Denker et al. 2008; Wessler and Kirkpatrick2008).To understand the origin and the diversification of the neu-ral cholinergic system, we studied AchE activity in a memberof the supposedly earliest offshoot of the Bilateria, the phylumAcoela. Molecular phylogenies indicate that the Acoela is thesister group of the remaining Bilateria (Ruiz-Trillo et al. 1999,2004; Halanych 2004; Sempere et al. 2007; Wallberg et al.2007; Hejnol et al. 2009). According to Wallberg et al. (2007)Acoela and Nemertodermatida are two early bilaterian clades.However, a recent phylogenomic analysis suggests, on thecontrary, the monophyletic nature of the Acoelomorpha(Hejnol et al. 2009). Obviously, this is still an unsettled issue.Nevertheless, Acoela probably represents the first group witha ‘centralized’ nervous system (Tyler and Hyra 1998; Reuteret al. 2001) or, more specifically, with a mass of neurons