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Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission

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Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission and play roles in many
cognitive processes. They are under intense research as potential targets of drugs used to treat neurodegenerative
diseases and neurological disorders such as Alzheimer's disease and schizophrenia. Invertebrate nAChRs are targets of anthelmintics as well as a major group of insecticides, the neonicotinoids. The honey bee, Apis mellifera, is one of the most beneficial insects worldwide, playing an important role in crop pollination, and is also a valuable model system for studies on social interaction, sensory processing, learning, and memory. We have used the A. mellifera genome information to characterize the complete honey bee nAChR gene family. Comparison with the fruit fly Drosophila melanogaster and the malaria mosquito Anopheles gambiae shows that the honey bee possesses the largest family of insect nAChR subunits to date (11 members). As with Drosophila and Anopheles, alternative splicing of conserved exons increases receptor diversity. Also, we show that in one honey bee nAChR subunit, six adenosine residues are targeted for RNA A-to-I editing, two of which are evolutionarily conserved in Drosophila melanogaster and Heliothis virescens orthologs, and that the extent of editing increases as the honey bee lifecycle progresses, serving to maximize receptor diversity at the adult stage. These findings on Apis mellifera enhance our understanding of nAChR functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.
The honey bee, Apis mellifera, is an important beneficial insect in agriculture. In addition to producing honey and
beeswax, the contribution of A. mellifera to crop pollination is valued at more than $14 billion dollars per year in
the U.S. alone (United States Department of Agriculture http://www.ars.usda.gov/main/main.htm). Honey bees live in societies of considerable complexity and thus are studied as models for social behavior (Robinson et al. 1997).

The neonicotinoids are the newest major group of insecticides, which includes acetamiprid, clothianidin, dinotefuran,
imidacloprid, nitenpyram, thiacloprid, and thiamethoxam (Tomizawa and Casida 2005). The worldwide annual sales of neonicotinoids amounts to 1 billion dollars, and they are used against piercing-sucking pests (aphids, leafhoppers, and white-flies) of major crops. In France, the use of imidacloprid has been suspended over concerns that it may be having a drastic effect on bee populations (http://www.pan-uk.org/press/pr140604.htm), highlighting the importance that effective insecticides should also show selectivity within insects so that pollinators such as A. mellifera are spared. While the link between imidacloprid use and bee population decline has yet to be proven, studies have shown that imidacloprid is highly toxic to A. mellifera (Suchail et al. 2004) and at sublethal doses can alter honey bee foraging and learning (Guez et al. 2001; Lambin et al. 2001; Decourtye et al. 2004). Neonicotinoids act as agonists on their molecular targets, nicotinic acetylcholine receptors (nAChRs) (Matsuda et al. 2001), which are prototypical members of the cys-loop ligand-gated ion channel (LGIC) superfamily (Karlin 2002). The fast actions of acetylcholine (ACh) at synapses are mediated by nAChRs, which consist of five homologous subunits arranged around a central ion channel (Corringer et al. 2000; Unwin 2005). Analyses of completed genomes have revealed diverse nAChR gene families with mammals possessing 16 subunit genes, chicken, 17 (Millar 2003), Fugu rubripes, 28 (Jones et al. 2003), and Caenorhabditis elegans, at least 27 (Jones and Sattelle 2004). In contrast, Drosophila melanogaster and Anopheles gambiae have notably smaller nAChR gene families, each consisting of 10 subunits (Jones et al. 2005; Sattelle et al. 2005).

To date, four A. mellifera nAChR subunits (Apis2, Apis3, Apis7-1, and Apis7-2) have been identified (Thany et al.
2003, 2005), which are expressed in brain structures that play roles in learning and memory, olfactory signal
processing, mechanosensory antennal input, and visual processing. These findings are consistent with ACh being a
major excitatory neurotransmitter in the insect nervous system (Breer and Sattelle 1987; Lee and O'Dowd 1999). Patch clamp studies have demonstrated the existence of a distinct nAChR subtype in the honey bee nervous system that is blocked by the nAChR antagonists -bungarotoxin (-Btx), dihydroxy--erythroidine and methyllycaconitine, while nicotine and imidacloprid acted as partial agonists on this receptor (Goldberg et al. 1999; D?glise et al. 2002; Wustenberg and Grunewald 2004). Another study has shown the presence of two nAChR populations that differ in their responses to imidacloprid but not ACh (Nauen et al. 2001). The involvement of nAChRs in honey bee behavior has also been investigated. Injection of the nAChR agonist, nicotine, showed that potentiation of the cholinergic system improves short term memory (Thany and Gauthier 2005) and injection of the nAChR antagonist, mecamylamine, inhibited olfactory learning or memory recall depending upon the site of injection (Lozano et al. 1996, 2001). Recently it has been demonstrated that one distinct nAChR sub-type, which is -Btx sensitive, is involved in long-term memory, whereas a second subtype, which is -Btx insensitive, but is affected by mecamylamine, plays a role in retrieval processes (Dacher et al. 2005). Interestingly, this mirrors to a certain extent the mammalian central nervous system, where there are two predominant nAChR subtypes, the 7 and 42 receptors, that are -Btx sensitive and insensitive, respectively, and both receptor subtypes play a role in memory (for review, see Hogg et al. 2003). Since individual nAChR subunits can confer distinct pharmacological properties on a receptor (Romanelli and Gualtieri 2003), the multiple nAChR subtypes present in the honey bee nervous system are likely to be determined by their subunit composition. Identifying the full complement of honey bee nAChR subunits represents a critical step in understanding the variety of roles played by nAChRs in the honey bee nervous system and the exquisite repertoire of bee behavior, as well as in identifying particular targets of chemical compounds. Here we have used the A. mellifera genome to describe the complete honey bee nAChR gene family.

Splice variants increase Apis nicotinic receptor diversity

Two Apis nAChR subunits, Amel4 and Amel6, have alternatively spliced exons most likely arising from tandem exon
duplication (Kondrashov and Koonin 2001). As with D4 and Agam4 (Lansdell and Millar 2000; Jones et al. 2005), Amel4 possesses two alternatives for exon 4 (denoted exon4 and exon4') (Fig. 4A). However, whereas D6 and Agam6 have two alternatives for exon 3 (Grauso et al. 2002; Jones et al. 2005), Amel6 has only a single exon. For 6 exon 8, both Apis and Anopheles have two alternatives, while Drosophila has three, although the mosquito possesses exons analogous to D6 8b and 8c (Jones et al. 2005), while the honey bee clearly possesses 8a and 8b-like exons.


We have used the available A. mellifera genome information to complete the characterization of the honey bee nAChR gene family, thus describing the first complete set of Hymenoptera nAChR subunits and the third insect nAChR gene family following those of the two Diptera, A. gambiae (Jones et al. 2005) and D. melanogaster (Sattelle et al. 2005). The three insect species represent 280 million years of evolution (Carpenter and Burnham 1985; De Gregorio and Lemaitre 2002) where the nAChR gene family has remained compact with A. mellifera having 11 genes encoding nAChR subunits, whereas both D. melanogaster and A. gambiae possess 10 genes (Jones et al. 2005; Sattelle et al. 2005). The nAChR subunit composition of Apis most closely resembles that of Anopheles in that both possess nine and one subunit, while Drosophila has seven and three . The extra honey bee subunit is a subunit (Amel2) making A. mellifera only the second insect known to possess more than one non- type subunit.

The characterization of the full complement of honey bee nAChR subunits presents an important basis for associating particular nAChR subtypes with key aspects of behavior, identifying receptor subtypes targeted by neonicotinoids as well as developing insecticides with improved selectivity. Indeed, comparison of complete insect nAChR gene families has identified a highly divergent subunit group (the D3 group) as well as species-specific proteome diversification arising from alternative splicing and RNA editing, all of which represent promising subunit differences to target for future rational insecticide design. While studies using heterologous expression systems such as Xenopus laevis oocytes have proven instructive in characterizing vertebrate nAChRs (Corringer et al. 2000) and low levels of functional expression of an insect subunit, L1, have been observed in Xenopus oocytes (Marshall et al. 1990), expression of functional insct nAChRs has so far proven elusive (Sattelle et al. 2005). Nevertheless, Drosophila nAChR subunits cn form robust functional channels when coexpressed with a vertebrate 2 subunit (Bertrand et al. 1994) and studies on such hybrid receptors have provided insights into the selectivity of neonicotinoids for insect nAChRs over those of vertebrates (Matsuda et al. 1998; Ihara et al. 2003), regions of subunit proteins involved in neonicotinoid interactions (Shimomura et al. 2002, 2003, 2004), and the actions of different neonicotinoids (Ihara et al. 2004). Also, computer models of insect nAChRs have been recently generated, which permit docking experiments to assess interactions with compounds of interest (Sattelle et al. 2005). Similar studies combining functional expression with molecular modeling of Apis nAChRs are likely to prove useful in screening for novel compounds that show low selectivity for honey bee receptors and in dissecting the mechanisms of insecticide actions and selectivity on nAChRs.


We are indebted to the A. mellifera Genome Project (Human Genome Sequencing Center), which provided the starting point for this study. We thank Sandrine Paute for technical support. We also thank Ryszard Maleszka and Chris Ponting for encouragement, support, and helpful comments on the manuscript.

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