Cannabinoid Receptors Are Absent in Insects JOHN MCPARTLAND, 1 * VINCENZO DI MARZO, 2 LUCIANO DE PETROCELLIS, 2 ALISON MERCER, 3 AND MICHELLE GLASS 4 1 GW Pharmaceuticals, Ltd., Porton Down Science Park, Salisbury, Wiltshire SP4 0JQ, United Kingdom 2 Endocannabinoid Research Group, CNR-Istituto per la Chimica di Molecole di Interesse Biologico (VDM) and Istituto di Cibernetica (LDP), Napoli 80072, Italy 3 Department of Zoology, University of Otago, Dunedin, New Zealand 4 Laboratory of Cell Biology, NIDCD, Rockville, Maryland 20850 ABSTRACT The endocannabinoid system exerts an important neuromodulatory role in mammals. Knockout mice lacking cannabinoid (CB) receptors exhibit significant morbidity. The endo- cannabinoid system also appears to be phylogenetically ancient—it occurs in mammals, birds, amphibians, fish, sea urchins, leeches, mussels, and even the most primitive animal with a nerve network, the Hydra. The presence of CB receptors, however, has not been examined in terrestrial invertebrates (or any member of the Ecdysozoa). Surprisingly, we found no specific binding of the synthetic CB ligands [ 3 H]CP55,940 and [ 3 H]SR141716A in a panel of insects: Apis mellifera, Drosophila melanogaster, Gerris marginatus, Spodoptera frugiperda, and Zophobas atratus. A lack of functional CB receptors was confirmed by the inability of tetrahydrocannabinol (THC) and HU210 to activate G-proteins in insect tissues, utilizing a guanosine-5'-O-(3-[ 35 ]thio)-triphosphate (GTPS) assay. No orthologs of human CB receptors were located in the Drosophila genome, nor did we find orthologs of fatty acid amide hydrolase. This loss of CB receptors appears to be unique in the field of comparative neurobiology. No other known mammalian neuroreceptor is understood to be missing in insects. We hypothesized that CB receptors were lost in insects because of a dearth of ligands; endogenous CB ligands are metabolites of arachidonic acid, and insects produce little or no arachidonic acid or endocannabinoid ligands, such as anandamide. J. Comp. Neurol. 436: 423– 429, 2001. © 2001 Wiley-Liss, Inc. Indexing terms: cannabinoid receptors; neurotransmitters; anandamide; tetrahydrocannabinol; GTPs; fatty acid amide hydrolase; sequencing analysis; insects; Cannabis The endocannabinoid system consists of a small family of G-protein-coupled receptors, endogenous ligands, and a ligand-metabolizing enzyme. Two cannabinoid (CB) recep- tors are currently known: CB1 predominates in the cen- tral nervous system, whereas CB2 is largely restricted to cells of immune function (Felder and Glass, 1998). The known ligands, arachidonylethanolamide (anandamide) and sn-2 arachidonylglycerol (2-AG), are derived from membrane phospholipids that contain arachidonate (Mechoulam et al., 1998). These ligands are metabolized by fatty acid amide hydrolase (FAAH), formerly known as anandamide amidohydrolase (Deutsch and Chin, 1993). In mammals, the endocannabinoid system exerts a sig- nificant neuromodulatory role (Di Marzo et al., 1998). Mice with a targeted deletion of CB1 receptors (CB1 -/- mice) exhibit altered nociceptive responses, extreme hy- pomobility, and significantly increased mortality (Zimmer et al., 1999). Human brains contain very high levels of CB1 receptors, about 10 times greater than opioid recep- tors (Sim et al., 1996). These findings illustrate the phys- iological importance of the endocannabinoid system. Moreover, the system appears to be phylogenetically ancient. Murphy et al. (2001) utilized the CB1 receptor gene in a phylogenetic analysis of mammals; they cloned Grant sponsor: GW Pharmaceuticals; Grant sponsor: MURST (Fondi strutturali). *Correspondence to: John McPartland, Faculty of Health & Environ- mental Science, UNITEC, Private Bag 92025, Auckland, New Zealand. E-mail: jmcpartland@unitec.ac.nz Received 11 April 2001; Revised 11 May 2001; Accepted 15 May 2001 THE JOURNAL OF COMPARATIVE NEUROLOGY 436:423– 429 (2001) © 2001 WILEY-LISS, INC.