Acetylcholinesterase genes within the Diptera: takeover and loss in true flies Elise Huchard, Michel Martinez, Haoues Alout, Emmanuel Douzery, Georges Lutfalla, Arnaud Berthomieu, Claire Berticat, Michel Raymond, Mylene Weill To cite this version: Elise Huchard, Michel Martinez, Haoues Alout, Emmanuel Douzery, Georges Lutfalla, et al.. Acetyl- cholinesterase genes within the Diptera: takeover and loss in true flies. Proceedings of the Royal Society B: Biological Sciences, Royal Society, The, 2006, 273 (1601), pp.2595-2604. 10.1098/rspb.2006.3621. hal-01945529 HAL Id: hal-01945529 https://hal.archives-ouvertes.fr/hal-01945529 Submitted on 29 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Proc. R. Soc. B (2006) 273, 2595–2604 doi:10.1098/rspb.2006.3621 Published online 18 July 2006 Acetylcholinesterase genes within the Diptera: takeover and loss in true flies Elise Huchard1, Michel Martinez2, Haoues Alout1, Emmanuel J. P. Douzery1, Georges Lutfalla3, Arnaud Berthomieu1, Claire Berticat1, Michel Raymond1,* and Myle`ne Weill1 1Institut des Sciences de l’Evolution (UMR 5554 CNRS), C.C. 065, and 3UMR 5124 CNRS, C.C.86, Universite´ de Montpellier II, 34095 Montpellier cedex 5, France 2INRA, Unite´ d’Ecologie animale et Zoologie agricole, 2, place Pierre Viala, 34060 Montpellier Cedex 01, France It has recently been reported that the synaptic acetylcholinesterase (AChE) in mosquitoes is encoded by the ace-1 gene, distinct and divergent from the ace-2 gene, which performs this function in Drosophila.Thisisan unprecedented situation within the Diptera order because both ace genes derive from an old duplication and are present in most insects and arthropods. Nevertheless, Drosophila possesses only the ace-2 gene. Thus, a secondary loss occurred during the evolution of Diptera, implying a vital function switch from one gene (ace-1) to the other (ace-2). We sampled 78 species, representing 50 families (27% of the Dipteran families) spread over all major subdivisions of the Diptera, and looked for ace-1 and ace-2 by systematic PCR screening to determine which taxonomic groups within the Diptera have this gene change. We show that this loss probably extends to all true flies (or Cyclorrhapha), a large monophyletic group of the Diptera. We also show that ace-2 plays a non-detectable role in the synaptic AChE in a lower Diptera species, suggesting that it has non-synaptic functions. A relative molecular evolution rate test showed that the intensity of purifying selection on ace-2 sequences is constant across the Diptera, irrespective of the presence or absence of ace-1, confirming the evolutionary importance of non-synaptic functions for this gene. We discuss the evolutionary scenarios for the takeover of ace-2 and the loss of ace-1, taking into account our limited knowledge of non-synaptic functions of ace genes and some specific adaptations of true flies. Keywords: acetylcholinesterase; evolution of gene function; Diptera; duplication; gene loss 1. INTRODUCTION ace-2 (e.g. in flies) encodes the main AChE, implying that The recent discovery of the gene coding of the synaptic ace-2 took over the function of ace-1 during evolution. acetylcholinesterase (AChE) in mosquitoes has led to a The synaptic AChE is involved in a vital function, and it paradox in their evolution. AChE stops neurotransmission is assumed that this function has always been present in the sensorial synapses of insects by hydrolysing the during the evolution of insects. Apparently, AChE has neurotransmitter acetylcholine (Toutant 1 989). The been naturally selected for its particularly high enzymatic Anopheles gambiae genome contains two ace genes: ace-1, activity. It is one of the fastest known enzymes with up to which encodes the main synaptic AChE (Weill et al. 2002) 104 substrate molecules being hydrolysed per second by and ace-2, which has an unknown function. These two each enzyme molecule, and its enzymatic velocity seems to genes have only 53% similarity at the amino acid level, and be limited only by the diffusion velocity of its substrate the overall ace phylogeny suggests that they diverged (Quinn 1987). This suggests that a slight reduction in its before the diversification of the arthropods (Weill et al. activity would somehow be translated to a significant 2002). Thus, both genes should be present in most fitness cost. In the mosquito Culex pipiens, a variant arthropods and have already been formally identified in synaptic AChE (coded by ace-1) differing by one amino Hemiptera, Hymenoptera, Lepidoptera and Acari acid (glycine 119 changed to serine, or G119S) is found in (Weill et al. 2002; Li & Han 2004; Russell et al. 2004; insecticide-treated areas. This variant is insensitive to Lee et al. 2006). By contrast, the Drosophila melanogaster some insecticides and has a 60% reduced activity, which is genome contains a single gene, ace-2 (Weill et al. 2002), associated with substantial fitness cost: about 11% per which encodes the synaptic AChE (Fournier et al. 1989). generation during the breeding season and 50–60% for The absence of ace-1 in the D. melanogaster genome can survival during the overwintering season (Lenormand therefore be explained by a secondary loss. Consequently, et al.1998; Lenormand et al. 1999; Lenormand & within the Diptera, either ace-1 (e.g. in mosquitoes) or Raymond 2000). The G119S mutation has also been detected in insecticide-resistant individuals in distant mosquito species (An. gambiae and An. albimanus, Weill * Author for correspondence ([email protected]). et al. 2004), suggesting that the AChE function cannot be greatly modified (to increase insensitivity to some The electronic supplementary material is available at http://dx.doi. org/10.1098/rspb.2006.3621 or via http://www.journals.royalsoc.ac. insecticides) without greatly affecting its optimal and uk. vital activity. Received 19 January 2006 2595 q 2006 The Royal Society Accepted 13 May 2006 2596 E. Huchard and others Gene takeover and loss in true flies Despite these physiological constraints, the gene (AJ748115); Schizaphis graminum (Q9BMJ1). All these ace-1 encoding the synaptic AChE has apparently changed sequences were used to design four pairs of primers: Moustdir within the Diptera, with the ancestral gene (ace-1) being (50 CCG-GGN-GCS-ACY-ATG-TGG-AA 30) with Mous- replaced by a divergent and distant gene (ace-2). This type trev (50 ACG-ATM-ACG-TTC-TCY-TCC-GA 30); P6dir of gene replacement, which pertains to a vital function, has (50ATM-GWG-TTY-GAG-TAC-ACS-GAY-TGG 30) with never been observed previously (to our knowledge), and P7rev (50 GGC-AAA-RTT-KGW-CCA-GTA-TCK-CAT thus we have no conceptual framework for understanding 30); Ace1dir3 (50 GAC-AAR-ATG-GTS-GGN-GAY- how such a phenomenon is possible and what sort of TAT-CA 30) with Ace1rev4 (50 CCR-TGC-ATM-ACR- selection has driven it. The Ao (aldehyde oxidase) gene in CCN-GTC-CA 30) or with P7rev. Polymerase chain reaction eukaryotes underwent a possibly related change; it derives (PCR) comprised 35 cycles of 93 8C for 30 s, 50 8C for 30 s neofunctionalization from a duplicate copy of Xdh and 72 8C for 30 s. These four pairs of primers were (xanthine dehydrogenase), and Xdh underwent a second systematically used for all samples. duplication in chordates. The new duplicated copy We amplified ace-2 using two pairs of degenerate primers: became a neofunctionalized Ao gene, with the first Ao ace2dir4 (50AAY-GCN-CCS-TGG-AGY-CAY-ATG-AC 30) gene subsequently disappearing from the vertebrate with ace2rev6 (50 CCV-GAR-TAS-GAR-TTC-CAY-TGY- genome (Rodriguez-Trelles et al. 2003). However, the TG 30), and ace2dir3 (50 TGG-ATY-TAY-GGB-GGY-GGS- Ao/Xdh and ace-1/ace-2 situations cannot be directly TTY-ATG 30) with ace2rev3 (50 GTC-ATR-TGR-CTC-C compared because the loss of ace-1 is not associated with AS-GGN-GCR-TT 30). These were designed by comparing anewace-2 copy, which could have acquired (by published ace-2 sequences from distant Diptera species: neofunctionalization) the same functions as ace-1. D. melanogaster (P07140), M. domestica (Q8MXC4), The first step in understanding this situation is to Lucilia cuprina (P91954), B. oleae (Q8MVZ4), An. gambiae determine which taxonomic groups within the Diptera (Q869C3) and C. pipiens (Q86GC8). The first pair of primers display this gene change. There are about 129 000 species was systematically used, with the second being used if the first described within the Diptera order, distributed among pair failed. 185 families (McAlpine & Wood 1989; Grimaldi & Engel PCR products were directly sequenced with an ABI prism 2005; plus update from M.M.). The main synaptic AChE 310 sequencer using the Big Dye Terminator kit. is encoded by ace-1 in Culex and Anopheles mosquitoes (Culicidae family), and by ace-2 in D. melanogaster. Both (c) Diptera phylogeny the housefly Musca domestica and the olive fruitfly The phylogenetic topology of the major taxonomic divisions Bactrocera oleae use ace-2 for their cholinergic synapses, in Diptera (infraorders or superfamilies illustrated in figure 1) as shown by ace-2 mutations providing insecticide and the identification of monophyletic groups were estab- resistance (Kozaki et al. 2001a; Walsh et al. 2001; Vontas lished according to published data (table 1; McAlpine & et al. 2002). Currently, there is no information available Wood 1989; Wiegmann 1993; Griffiths 1994; Cumming et al.