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Characterization of the nicotinic acetylcholine receptor ...scott. Receptor Subunit Gene Md a 2 om the Hr F ouse F, yl Musca domestica Jian-Rong Gao, Juliane M. Deacutis, and Jeffrey

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  • 30 Gao et al.

    Archives of Insect Biochemistry and Physiology January 2007 doi: 10.1002/arch.

    Archives of Insect Biochemistry and Physiology 64:3042 (2007)

    © 2006 Wiley-Liss, Inc. DOI: 10.1002/arch.20158 Published online in Wiley InterScience (www.interscience.wiley.com)

    Characterization of the Nicotinic Acetylcholine Receptor Subunit Gene Mda2 From the House Fly, Musca domestica

    Jian-Rong Gao, Juliane M. Deacutis, and Jeffrey G. Scott*

    A nicotinic acetylcholine receptor (nAChR) subunit gene, Mda2, was isolated and characterized from the house fly, Musca domestica. This is the first nAChR family member cloned from house flies. Mda2 had a cDNA of 2,607 bp, which included a 696 bp 5¢-untranslated region (UTR), an open reading frame of 1,692 bp, and a 219 bp 3¢-UTR. Its deduced amino acid sequence possesses the typical characteristics of nAChRs. Mda2 genomic sequence was 11.2 kb in length in the aabys strain and 10.9 kb in the OCR strain, including eight exons and seven introns. Based on the deduced amino acid sequence, Mda2 had the closest phylogenetic relationship to the Drosophila melanogaster Da2 and Anopheles gambiae Agama2, and a similar genomic structure to Da2. Quantitative real-time PCR analysis showed that Mda2 is expressed in the head and the thorax at 150- and 8.5-fold higher levels than in the abdomen. Linkage analysis of a Mda2 polymorphism indicates this gene is on autosome 2. The importance of these results in understanding the diversity and phylogenetic relationships of insect nAChRs, the physiology of nAChRs in the house fly, and the utility of nAChR sequences in resistance detection/monitoring is discussed. Arch. Insect Biochem. Physiol. 64:3042, 2007. © 2006 Wiley-Liss, Inc.

    KEYWORDS: nicotinic acetylcholine receptor; alpha subunit; genomic organization; quantitative real-time PCR;

    linkage analysis; Insecta; RNA editing

    Department of Entomology, Comstock Hall, Cornell University, Ithaca, New York

    Contract grant sponsor: Elanco Animal Health; Contract grant sponsor: Daljit S. and Elaine Sarkaria Professorship; Contract grant sponsor: Cornell Presidential

    Research Scholars Program.

    *Correspondence to: Dr. Jeffrey G. Scott, Department of Entomology, Comstock Hall, Cornell University, Ithaca, NY 14853. E-mail: [email protected]

    Received 13 June 2006; Accepted 28 September 2006

    INTRODUCTION

    Nicotinic acetylcholine receptors (nAChR) be-

    long to the Cys-loop superfamily of ligand-gated

    ion channels that include g-aminobutyric acid

    (GABA)-gated channels, glycine receptors, gluta-

    mate-gated Cl channels, and 5-hydroxytryptamine

    type 3 (5-HT3) receptors (Lester et al., 2004). The

    nAChRs play an essential role in the fast excita-

    tory neurotransmission at cholinergic synapses in

    the insect central nervous system (CNS) (Gundel-

    finger and Schulz, 2000) and are also the target

    site for the economically important neonicotinoid

    (including imidacloprid) insecticides (Narahashi

    1996; Tomizawa et al., 1999), as well as cartap and

    bensultap (Lee et al., 2003).

    The nAChRs are composed of five homologous

    subunits, typically 2 a and 3 non-a, but receptors

    consisting of only a subunits are also known (Cou-

    turier et al., 1990; Marshall et al., 1990). The a

    subunits are characterized by the presence of two

    adjacent cysteine residues in loop C, while the non-

    a subunits lack this cysteine doublet. Each sub-

    unit possesses a large N-terminal extracellular

    domain that includes the acetylcholine (ACh)

    binding site and four transmembrane domains

  • Characterization of Mda2 From M. domestica 31

    Archives of Insect Biochemistry and Physiology January 2007 doi: 10.1002/arch.

    (TM14) with TM2 contributing most of the amino

    acids that line the ion channel (Karlin, 2002). The

    ACh binding site, in native and functional recep-

    tors, is located at the interface of two subunits, and

    possibly consists of three loops (loops AC) of one

    subunit and by three (loops DF) of the other

    (Grutter and Changeux, 2001).

    Information on the subunit composition of a

    native nAChR of insects and their interaction with

    neonicotinoid insecticides is limited. Using neonico-

    tinoid-agarose affinity chromatography and immu-

    noprecipitation, the nAChR subunits ALS, Da2 (also

    known as second alpha-like Drosophila nAChR sub-

    unit, SAD) and SBD in Drosophila melanogaster (and

    their homologs in Musca) seem to assemble into

    an integral receptor (Chamaon et al., 2002; Tomi-

    zawa et al., 1996). Voltage-clamp electrophysiology

    studies revealed that Da2/chicken b2 receptors ex-

    pressed in Xenopus oocytes were highly sensitive

    to the actions of neonicotinoids, including imi-

    dacloprid, whereas imidacloprid had little effect on

    the ALS/chicken b2 receptors (Ihara et al., 2003,

    2004). A P242E substitution in Da2 was found to

    significantly reduce (approximately 5-fold) imi-

    dacloprid sensitivity in the Da2/chicken b2 nAChR

    (Shimomura et al., 2004, 2005), suggesting that

    Da2 is important in neonicotinoid toxicity to in-

    sects. However, the utility of using subunits from

    different species to assemble functional receptors

    has been questioned (Tomizawa et al., 2005). Re-

    cently, a Y151S mutation was identified in two

    nAChR subunits (a1 and a3) from imidacloprid-

    resistant brown planthoppers, Nilaparvata lugens,

    and was correlated with a 100-fold reduction in

    imidacloprid binding (Liu et al., 2005).

    Based on completed genome sequences, there are

    ten nAChR subunit genes in both D. melanogaster

    (Sattelle et al., 2005) and Anopheles gambiae (Jones

    et al., 2005). However, there is no complete gene

    sequence of any nAChR subunit from the house fly

    (Musca domestica L.), an important vector of human

    and animal diseases (Burgess, 1990). In 2004,

    imidacloprid was first registered for house fly con-

    trol in the United States and it is becoming widely

    used for this purpose, especially because of resis-

    tance to other insecticides that are registered against

    this pest (Darbro and Mullens 2004; Hamm et al.,

    2005; Kaufman et al., 2001). In order to develop

    neonicotinoid resistance monitoring/detection tools

    for use in house flies, and to further our understand-

    ing of the action of neonicotinoid insecticides, more

    information on the nAChRs is needed. In this study,

    we report the cDNA sequence, genomic organiza-

    tion, expression, and chromosomal linkage of the

    house fly nAChR subunit gene Mda2, the ortholog

    of Da2 from Drosophila.

    MATERIALS AND METHODS

    House Flies

    Three strains of house flies were used: aabys (in-

    secticide susceptible strain, with the recessive mor-

    phological markers ali-curve [ac], aristapedia [ar],

    brown body [bwb], yellow eyes [ye], and snipped wings

    [snp] on autosomes 1, 2, 3, 4, and 5, respectively),

    OCR (cyclodiene resistant), and Sullivan (field

    population collected from Sullivan County, New

    York in 2004). Flies were maintained in the labo-

    ratory as previously described (Scott et al., 2000).

    RNA Isolation and RT-PCR

    Messenger RNA was isolated from the heads of

    adult flies (

  • 32 Gao et al.

    Archives of Insect Biochemistry and Physiology January 2007 doi: 10.1002/arch.

    rification kit (Qiagen, Valencia, CA) and sequenced

    at the Cornell Biotechnology Resource Center.

    Rapid Amplification of cDNA Ends (RACE)

    The 3¢ and 5¢ RACE were performed using

    SMART RACE cDNA amplification kit (BD Bio-

    sciences, Palo Alto, CA). The 3¢- and 5¢-RACE-ready

    cDNA was synthesized with 1 mg of mRNA. The

    Ma2RaceF (5¢-TGGTGGTTCTACTTGCCCGCCGAT

    TCG-3¢) and Ma2RaceR (5¢-CGAATCGGCGGGCAA

    GTAGAACCACCA-3¢) primers were used (in con-

    junction with primers provided in the kit) for the

    3¢ and 5¢ RACE, respectively, according to the

    manufacturers instructions. The following thermal

    cycler program was used: 1 cycle of 95°C for 1 min,

    35 cycles of 95°C for 30 s, 68°C for 30 s, and 72°C

    for 3 min, and a final extension at 72°C for 10

    min. PCR product was analyzed on 1% agarose gel,

    purified with QIAEX® II gel extraction kit (QIAGEN

    Sciences, MD) and subsequently cloned into pCR®

    2.1-TOPO® vector and transformed into TOP10

    cells using TOPO-TA Cloning® kit (Invitrogen,

    Carlsbad, CA). DNA sequencing was performed at

    the Cornell Biotechnology Resource Center.

    Cloning of the Open Reading Frame (ORF)

    A fragment containing the ORF of Mda2 was

    amplified from the 5¢-RACE-ready cDNA with a for-

    ward primer Ma2F1 (5¢-AGCGCATCAGTTACG

    ACGTCACA-3¢) and a reverse primer Ma2R1 (5¢-

    CAGACTTGACATTTGTTAACATTCGAGGTG-3¢) us-

    ing Advantage® 2 polymerase mix (BD Biosciences

    Clontech). The PCR thermal program was 1 cycle

    of 95°C for 1 min, 30 cycles of 95°C for 30 s, 63°C

    for 30 s, and 68°C for 2.5 min and a final exten-

    sion at 68°C for 7 min. PCR product analysis, pu-

    rification, cloning, and sequencing were done as

    described above. Five clones were fully sequenced.

    Genomic DNA

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