Download pdf - Characterization of the nicotinic acetylcholine receptor ...scott.entomology.cornell.edu/136.pdfReceptor Subunit Gene Md a 2 om the Hr F ouse F, yl Musca domestica Jian-Rong Gao, Juliane

30 Gao et al.

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

Archives of Insect Biochemistry and Physiology 64:30�42 (2007)

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

Characterization of the Nicotinic AcetylcholineReceptor 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, Muscadomestica. This is the first nAChR family member cloned from house flies. Mda2 had a cDNA of 2,607 bp, which included a696 bp 5¢-untranslated region (UTR), an open reading frame of 1,692 bp, and a 219 bp 3¢-UTR. Its deduced amino acidsequence possesses the typical characteristics of nAChRs. Mda2 genomic sequence was 11.2 kb in length in the aabys strainand 10.9 kb in the OCR strain, including eight exons and seven introns. Based on the deduced amino acid sequence, Mda2had the closest phylogenetic relationship to the Drosophila melanogaster Da2 and Anopheles gambiae Agama2, and asimilar genomic structure to Da2. Quantitative real-time PCR analysis showed that Mda2 is expressed in the head and thethorax at 150- and 8.5-fold higher levels than in the abdomen. Linkage analysis of a Mda2 polymorphism indicates thisgene is on autosome 2. The importance of these results in understanding the diversity and phylogenetic relationships of insectnAChRs, the physiology of nAChRs in the house fly, and the utility of nAChR sequences in resistance detection/monitoring isdiscussed. Arch. Insect Biochem. Physiol. 64:30�42, 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


(TM1�4) 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 A�C) of one

subunit and by three (loops D�F) 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 (<1 d old) using QuickPrep� micro

mRNA purification kit (Amersham Biosciences,

Piscataway, NJ). First-strand cDNA was synthesized

from 450 ng of mRNA by priming with oligo(dT)

using SuperScript� III first-strand synthesis system

for RT PCR kit (Invitrogen, Carlsbad, CA) accord-ing to the manufacturer�s instructions. A forwardprimer Ma2F (5¢-ACCCTGCGCCGCAAGACCCTCT-

3¢) and reverse primer Ma2R (5¢-CAGGGCCAGTGAGGTGGAGGGTATGA-3¢) were designed basedon the partial Mda2 sequence previously reported(Sgard et al., 1993). A 198-bp fragment was ampli-fied from the aabys strain using Advantage® 2 poly-merase mix (BD Biosciences Clontech, Palo Alto,CA) in a total volume of 25 ml. The PCR thermalprogram consisted of 1 cycle of 95°C for 1 min, 35

cycles of 95°C for 30 s, 64°C for 30 s, and 68°C for3 min and a final extension at 68°C for 10 min.

PCR product was purified using QIAquick PCR pu-

32 Gao et al.


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

manufacturer�s 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 Extraction, Fragment

Amplification, and Sequencing

Genomic DNA was extracted from individual

male house flies using the quick fly genomic DNA

prep method (www.fruitfly.org). Briefly, a male fly

was homogenized in 400 ml of buffer A (100 mM

Tris-HCl, pH 7.5, 100 mM EDTA, 100 mM NaCl,

and 0.5% SDS). The homogenate was incubated

at 65°C for 30 min, followed by 10-min incuba-

tion on ice after being mixed with 0.8 ml of LiCl/

KAc solution (4.3 M LiCl and 1.43 M KAc). The

mixture was centrifuged at 14,000g for 15 min at

25°C. DNA was precipitated from the supernatant

by addition of isopropanol, and then pelleted by

centrifugation at 14,000g for 15 min at 25°C. The

DNA pellet was washed with 70% ethanol and dis-

solved in 150 ml of TE buffer.

To study the genomic organization of Mda2,

four genomic fragments were amplified using the

Advantage® 2 polymerase mix (BD Biosciences

Clontech) with primer pairs: gM2IF1 (5¢-GGTGCA

CAGGACAGCGGAGAG-3¢) and M2RU (5¢-GCA

CAACCATTTGAAGTGGGACCA-3¢), gM2IIIF1 (5¢-

AAGGATCAGATTCTAACCACAAACGTGT-3¢) and

gM2VIR2 (5¢-ACATGCCGACGGTGGGAATGATCAG-

3¢), gM2VF1 (5¢-GACAAGGATAACAAGGTAGAGA

TCGGCA-3¢) and gM2VIIR1 (5¢-TCGTCCTGGCGC

TGCATATGGT-3¢), gM2VIIF1 (5¢-GCACCTTGAGC

GGCTACAAC-3¢), and gM23UR (5¢-CAGAGCGTT

GAGTGAGACTTGACATTTG-3¢). The PCR programs

consisted of 1 cycle of 95°C for 2 min, 32 cycles

of 95°C for 30 s, 64 to 65°C for 30 s, and 68°C for

2 to 5 min and a final extension at 68°C for 10

min. PCR product analysis, purification, cloning,

and sequencing were done as described above. Two

or three clones were fully sequenced for each am-

plification. The four overlapping sequences were

aligned to get the entire genomic sequence.

Sequence Analysis

The signal peptide was predicted by SignalP

(Nielsen et al., 1997). The phosphorylation sites

and N-glycosylation sites were identified by the

PROSITE database (Falquet et al., 2002). Multiple

sequence alignment was performed with CLUSTAL

W (Megalign program, DNASTAR Inc., Madison,

WI). The phylogenetic analysis was performed us-

ing the PHYLIP software package (Felsenstein,

1993). The phylogenetic tree was constructed by



the neighbor-joining method (Saitou and Nei,

1987). Bootstrap values were calculated with

SEQBOOT program (Felsenstein, 1985) on 1,000

replications. The phylogenetic tree was drawn by the

TreeView program (Page, 1996). The cDNA and ge-

nomic DNA sequences of Mda2 were deposited in

GenBank (accession nos: DQ372062, DQ372063,

and DQ372064). Genomic and cDNA sequence

comparison was performed using SIM4 software

(Florea et al., 1998).

Quantitative Real Time RT-PCR

Total RNA was isolated from heads, thoraces,

and abdomens of aabys flies (3�24 h post-emer-

gence) using TRIzol® reagent (Invitrogen, Carlsbad,

CA) and treated with DNase (Ambion, Austin, TX).

First-strand cDNA was synthesized in a total 100-

ml reaction volume using 18 mg of the DNase-

treated RNA with TaqMan Reverse transcription

reagents using random hexamers (Applied Bio-

systems, Foster City, CA). PCR (20 ml per reaction)

was performed using 2 ml of the cDNA samples,

SYBR Green PCR core reagents (Applied Bio-

systems) and specific primers qMa2F1 (5¢-TGTGCC

TCCTAATGCTACTAATCCT-3¢) and qMa2R1 (5¢-

TCGTAGAGTCGTTTCGCATCTG-3¢) using an ABI

PRISM 7900 HT Sequence Detection System with

Sequence Detection Software (version 2.1) (Applied

Biosystems). All the procedures were conducted ac-

cording to the manufacturer�s instructions. The PCR

program consisted of 50°C for 2 min and 95°C

for 10 min for initiation, 40 cycles of 95°C for 15

s and 60°C for 1 min, followed by 95°C for 15 s,

60°C for 15 s, and 95°C for 15 s for melting curve

analysis. The PCR specific amplification was as-

sessed by the melting curve analysis and electro-

phoresis of the PCR products on 1.8% agarose gel.

External standard curves were constructed using 8

serial 5-fold dilutions of plasmids (pCR® 2.1 con-

taining Mda2 ORF) starting from 0.04 ng/ml. This

analysis was replicated five times. The gene expres-

sion levels were analyzed with a randomized com-

plete block design using a one-way ANOVA. Gene

copy numbers were transformed to log values and

significant differences were determined using

Tukey�s test at P = 0.05 (PROC GLM, SAS Insti-

tute, 2001).

Linkage Analysis

Linkage analysis was performed by the associa-

tion of Mda2 polymorphisms (between aabys and

OCR) with the five mutant markers of the aabys

strain (Kozaki et al., 2002). Female aabys were

crossed with male OCR to produce F1 flies het-

erozygous for all five autosomes. The F1 males

were then backcrossed to the homozygous aabys

females. The offspring were sorted according to

phenotype. Five phenotypes were used to conduct

the linkage analysis, being heterozygous at only

one chromosome, as indicated by the absence of

a recessive morphological marker. Flies that were

heterozygous for each of autosomes one through

five were denoted as +;ar;bwb;ye;snp, ac;+;bwb;

ye;snp, ac;ar;+;ye;snp, ac;ar;bwb;+;snp, ac;ar;bwb;ye;+,

respectively.

Genomic DNA was extracted as previously

described from 12 aabys and 19 OCR parental in-

dividuals, 10 F1 individuals, and at least three in-

dividuals from each previously stated backcross

genotypes. Genomic DNA fragments (1,379 bp from

aabys and 1,362 bp from OCR) were amplified us-

ing the Advantage® 2 polymerase mix (BD Bio-

science Clontech) with a forward primer (gM2VIIF1:

5¢-GCACCTTGAGCGGCTACAAC-3¢) and a reverse

primer (gM2VIIIR2: 5¢-GACGGAGCCTCGCCCAGTATC-3¢) using the following thermal cycler pro-gram: 1 cycle of 95°C for 1 min, 30 cycles of 95°Cfor 30 s, 64°C for 30 s and 72°C for 2 min, and afinal extension at 72°C for 7 min. The PCR prod-uct was purified using QIAquick PCR purificationkit (Qiagen, Valencia, CA) and then sequenced us-ing another primer (mdnachra2f2: 5¢-AAGCAATCACGGCAAGGGCATC-3¢).

RESULTS AND DISCUSSION

Cloning of the nAChR Subunit Mda2 cDNA

A 198-bp fragment was amplified from aabysflies using a pair of Mda2 primers designed based

on the partial sequence reported by Sgard et al.

34 Gao et al.


(1993). Based on the sequence of this fragment,

two primers (Ma2RaceF and Ma2RaceR) were syn-

thesized for the 3¢ and 5¢RACE. The 5¢RACE and

3¢RACE yielded a 1,536-bp and 1,097-bp product,

respectively. These fragments partially overlapped

the original 198-bp fragment. Primers were then

designed to amplify a fragment (2,034-bp) con-

taining the entire open reading frame (ORF). The

complete cDNA consists of 2,607 nucleotides with

an ORF of 1,689 nucleotides encoding 563 amino

acid residues (Fig. 1). The cDNA sequence includes

the start codon ATG at position 697�699 and the

stop codon TAA at position 2,386�2,388. The 696-

bp 5¢-untranslated region (UTR) contains 7 in-

frame stop codons, indicating that the ATG at

697�699 is the correct start codon. The 222-bp 3¢

UTR contains a eukaryotic consensus polyadenyl-

ation signal AATAAA at position 2,529�2,534 and

a poly(A)26 tail. There were 12 single nucleotide

polymorphic (SNP) sites identified within the ORF

of the sequenced clones, but only one (A1494G)

of them resulted in amino acid change (I266M).

The I266M substitution is located in the middle

of the conserved transmembrane 1 domain and

might affect the function of the ion channel.

The Mda2 cDNA encodes a 563-aa peptide that

possesses a predicted (by SignalP) 32-aa signal pep-

tide. The mature protein has a calculated molecu-

lar weight of 60.7 kDa and isoelectric point of 6.04.

The molecular weight is similar to that estimated

for native nAChRs of house fly heads (61�69 kDa)

determined by LiDS-PAGE analysis of samples of

neonicotinoid-agarose affinity and alpha-bungaro-

toxin affinity chromatography (Tomizawa et al.,

1996). The mature protein has 15 potential phos-

phorylation sites for protein kinase C, casein kinase

II, tyrosine kinase, and cAMP- and cGMP-depen-

dent protein kinase, five of which are located in

the N-terminal extracellular domain, eight located

in the cytoplasmic TM3-TM4 linker, and two lo-

cated in the C-terminal extracellular domain. Phos-

phorylation is important in regulating receptor

gene expression, as well as in altering desensitiza-

tion and recovery from desensitization (Char-

pantier et al., 2005; Courjaret et al., 2003; Fenster

et al., 1999; Wecker et al., 2001). The protein has 5

potential N-glycosylation sites that may be involved

in receptor assembly, ligand binding, regulation of

receptor desensitization, and ion permeability

(Chen et al., 1998; Nishizaki 2003; Wanamaker

and Green, 2005).

Alignment of the house fly deduced amino acid

sequence with Drosophila Da2 and Anopheles

Agama2 showed that Mda2 possesses typical

nAChR a subunit characteristics (Fig. 2). The pro-

tein includes the long N-terminal extracellular do-

main and the four hydrophobic transmembrane

domains (TM1-4), which are conserved in nAChRs

(Le Novere and Changeux, 1995). The N-terminal

domain contains the characteristic two cysteines

separated by thirteen residues (i.e., cysteine loop,

found in all ligand-gated ion channels) (Karlin,

2002), the ACh-binding-site-forming regions (loops

A-F) (Grutter and Changeux, 2001), and the YXCC

motif in loop C, the signature of a subunit (Kao

et al., 1984).

Phylogenetic Analysis and Sequence Comparison

The entire set of the nAChR family was ana-

lyzed from both D. melanogaster and An. gambiae

by taking advantage of the completed genome se-

quences. Although both of them have 10 subunit

genes, their components are different. D. melano-

gaster consists of 7 a and 3 non-a subunits while

An. gambiae consists of 9 a and 1 non-a (Jones et

al., 2005; Littleton and Ganetzky, 2000). To exam-

ine the evolutionary relationship between Mda2

and the nAChR subunit genes of Drosophila and

Anopheles (where complete genome sequences are

available), we conducted a phylogenetic analysis

using the neighbor-joining method. Results of this

analysis show that Mda2 is most closely related to

Da2 and Agama2 with these three a2 subunits

clustering together (Fig. 3). A BLASTP search, per-

formed using the NCBI database, showed that

Mda2 was most similar to six other insect nAChR

subunit genes: Da2/SAD (GenBank accession no.

P17644) from D. melanogaster, Agama2 (AAU

12504) from An. gambiae, a2 (AAD09808) from

Heliothis virescens, aL1 (CAA39081) from Schisto-

cerca gregaria, Apisa2 (NP_001011625) from Apis



Fig. 1. Nucleotide and deduced amino acid sequences

of Mda2 cDNA from aabys house flies (GenBank acces-

sion no. DQ372062). The translation start codon, ATG, is

bolded. The stop codon, TAA, is underlined with a star.

Bold-faced nucleotides and bold-faced amino acids in-

dicate sites of polymorphisms and related amino acid

changes. The predicted signal peptide cleavage site is marked

with a vertical arrow. The potential N-linked glycosylation

sites are underlined. The potential phosphorylation sites

are boxed. The stop codons upstream of the coding region

are bolded. A putative polyadenylation signal, AATAAA, in

the 3¢-untranslated region is bolded. The primers used to

amplify the ORF are bolded and underlined.

36 Gao et al.


Fig. 2. Alignment of the deduced amino acid sequence

of Mda2 (accession no. DQ372062) from aabys house

flies, with the homologous genes of Drosophila melanogaster

(P17644) and Anopheles gambiae (AAU12504). Non-con-

served amino acid residues in at least two of the sequences

are shaded with black. Signal peptide (SP) and transmem-

brane domains (TM1�4) are underlined. The location of

six loops (loop A�F) proposed to be important in form-

ing the agonist/antagonist binding site are also indicated.

The cysteine doublet (characteristics of nAChR a subunits)

is marked with two asterisks.

mellifera and Mpa1 (CAA57476) from Myzus

persicae. Mda2 shares 88.54, 84.0, 78.9, 76.3, 76.1,

and 67.0 % identity, respectively, based on pairwise

amino acid sequence comparisons. Therefore, the

cloned nAChR subunit gene was named Mda2.

Genomic Organization and Sequence Comparison

To determine the genomic organization of

Mda2, an 11,115-bp region (GenBank accession no.

DQ372063) where Mda2 resides was PCR-ampli-

fied using aabys genomic DNA as the template. By

comparing it with the Mda2 cDNA sequence, this

11.1-kb fragment was determined to contain 8 ex-

ons interrupted by 7 introns. The exons ranged

from 155�635 bp in length, while the introns

ranged from 65 to 6,929 bp (Fig. 4). All the splice

sites (intron/exon boundaries) conformed to the

GT-AG rule (Table 1) (Mount et al., 1992). The

start codon is located in exon II and the stop codon

in exon VIII. The N-terminal extracellular domain

was assembled from exons II�VI. The transmem-

brane domains TM1�3 are located in exon VI while

TM4 is in exon VIII. The exons from the genomic

sequence were compared with the cDNA sequence

and only one nucleotide (A at position 1494, Fig.

1) differed. Because this change (A1494G) resulted

in amino acid substitution (I266M), we checked



Fig. 3. Evolutionary relationships of deduced amino acid

sequences of Mda2 (GenBank accession no. DQ372062)

with nAChR subunits of Drosophila and Anopheles con-

structed by the neighbor-joining method. Bootstrap val-

ues with 1,000 trials are indicated on branches. The scale

bar represents substitutions per site. Sequences used: Droso-

phila subunits: Da1, GenBank accession no. CAA30172;

Da2, CAA36517; Da3, CAA75688; Da4, CAB77445; Da5,

AAM13390; Da6, AAM13393; Da7, CAD86936; Db1,

CAA27641; Db2, CAA39211; Db3, CAC48166; Anopheles

subunits: Agama1, AAU12503, Agama2, AAU12504,

Agama3, AAU12505, Agama4, AAU12506, Agama5,

AAU12508, Agama6, AAU12509, Agama7, AAU12511,

Agama8, AAU12512, Agama9, AAU12513, Agamb1,

AAU12514.

13 partial genomic DNA sequences amplified from

5 aabys, 5 OCR, and 3 Sullivan flies. Interestingly,

only A was present at this position in all of the

sequences. However, in our cloned cDNA se-

quences, 67% (4/6) are G and 33% (2/6) are A.

This result suggests that the polymorphism at 1490

is likely due to the A-to-I RNA editing (Maas et

al., 2003; Seeburg, 2002; Simpson and Emeson

1996). Clarification of this putative editing in

Mda2 will require further study. A-to-I RNA edit-

ing was previously found in Drosophila Da5, Da6,

Db1, and Db2 subunit genes (Grauso et al., 2002;

Hoopengardner et al., 2003), although the physi-

ological significance of the nAChR editing is not

38 Gao et al.


well understood. RNA editing of an insect sodium

channel (BgNav) is carried out in a tissue- and de-

velopment-specific manner, and results in channels

with different gating properties (Song et al., 2004).

A similar role for RNA editing of nAChRs is pos-

sible.

The genomic sequence of Mda2 was next de-

termined from OCR house flies. It consisted of

10,889 bp (GenBank accession no. DQ372064),

slightly shorter than that of aabys flies. The sizes

of exons I to VIII are 281, 635, 155, 181, 160, 574,

202, and >327 bp, respectively. The sequence of

exon VIII in OCR is shorter than that of aabys be-

cause the reverse primer used in PCR amplifica-

tion is located 61 bp upstream of the cDNA 3¢end.

The sizes of introns I to VII are 302, 6,680, 66, 66,

67, 105, and 1,088 bp, respectively. The most sig-

nificant sequence difference of OCR was detected

in intron II where a 192- and 36-bp deletion oc-

curred compared to that of aabys flies. Compari-

son of the deduced amino acid sequences of Mda2

from the two strains revealed the presence of eight

SNPs. However, none of these resulted in amino

acid changes.

The overall organization of Mda2 and Da2 are

strikingly similar (Fig. 4), with each gene having

eight exons. The most notable differences were the

lengths of exon II, intron II, and intron IV (Fig.

4). Based on the incomplete cDNA (accession no.

AY705395) and genomic sequences (AAAB

01008859) of Agama2, some of the structure of

Agama2 cannot be determined. The major differ-

ence between Mda2 (or Da2) and Agama2 is the

presence of two more exons in Agama2 (Jones et

al., 2005).

Gene Expression in Head, Thorax, and Abdomen

The Mda2 gene expression pattern in different

body parts was investigated using quantitative real-

time PCR. Mda2 expression was 150- and 8.5-fold

higher in the fly head and thorax, respectively, than

in the abdomen (Fig. 5). This pattern is consistent

with the idea that Mda2 is expressed in the CNS

of house flies. The CNS of the adult house fly is

highly specialized, consisting of a cephalic ganglion

(a complex of brain and suboesophageal ganglia)

in the head, and a thoracic compound ganglion

(a fusion of all the thoracic and abdominal gan-

glia) in the thorax (Hewitt, 1914). Indeed, this ex-

pression pattern agrees quite well with the previous

report of Jonas et al. (2003) who found that Droso-

phila Da2 transcripts and protein were expressed

exclusively in the CNS detected by in situ hybrid-

Fig. 4. Genomic structure of the Musca domestica Mda2

gene (GenBank accession no. DQ372062) and the Droso-

phila melanogaster Da2. Boxes and lines represent exons

and introns, respectively. Shaded boxes represent the open

reading frame. The sizes of exons are indicated above the

boxes and the sizes of introns are indicated under the lines.

The cDNA and genomic sequences of Da2 were obtained

from GenBank (accession nos. X53853 and AE003748).

TABLE 1. Exon-Intron Boundaries of the Housefly Mda2 Gene

cDNA

Intron splicing site Donor splice site Acceptor splice site

I 286�287 ACATTCTAAGgtactacacg cgcctttaagATGGGAGCTC

II 921�922 GATCGATTTGgtaagttaac cttttttcagAATTTAAAGG

III 1076�1077 TCTACAACAAgtaaatatat tttgtttcagTGCCGACGGC

IV 1257�1258 CGGTGACCAGgtaagtcgtc acccgcctagATCGATTTGA

V 1417�1418 CCGTATCCYGgtaagcaaaa cccatttcagACATATTCTT

VI 1991�1992 CCACGAATAGgtgagttccg atatttacagATTTAGTGGC

VII 2193�2194 ATTCAATGCGgtgagtwrga gtcatttcagGAAGATCAAG



ization and immunohistochemistry and suggested

that �the Da2 protein is a subunit of a synaptic

nicotinic receptor.� A recent study on the honey-

bee Apisa2 expression in the pupal and adult brains

showed that the Apisa2 transcripts were detected

in neurons including the dorsal and antennal

lobes, the calyces of mushroom bodies, the non-

compact Kenyon cells, outer compact Kenyon cells,

and the fenestrated cell layers between the lamina

and the medulla (Thany et al., 2005).

Linkage Analysis

Two Mda2 alleles (allele A, accession no. DQ

393143 and allele B, accession no. DQ393144, with

frequencies of 0.833 and 0.167, respectively) were

identified in the aabys strain and two alleles (al-

lele B and allele C, accession no. DQ372064, with

frequencies of 0.132 and 0.868, respectively) were

found in the OCR strain. If an individual homozy-

gous for the A allele was detected in one of the

five genotypes isolated from the backcross, this

indicated that Mda2 was not linked to the auto-

some having the wildtype trait. For example, if

an individual homozygous for the allele A had

the genotype +/ac;ar/ar;bwb/bwb;ye/ye;snp/snp, we

would conclude that Mda2 is not on autosome

1. Our initial analyses were done on three indi-

viduals for each of the backcross genotypes (hav-

ing mutant markers for four of the five auto-

somes). Individuals homozygous for allele A were

detected in all of the backcross genotypes except

for ac/ac;+/ar;bwb/bwb;ye/ye;snp/snp individuals

(Table 2). We confirmed the lack of allele A ho-

mozygotes in an additional seven individuals of

this genotype (Table 2). The lack of A allele ho-

mozygotes in the ac/ac;+/ar;bwb/bwb;ye/ye;snp/snp

individuals (Table 2) indicates that Mda2 is on

autosome 2. These results are in agreement with

Drosophila/Musca homology maps (Foster et al.,

1981). Male and female flies having each of the

mutant markers were observed in the backcross

(data not shown) indicating that sex determina-

tion in OCR is likely controlled by the Y chro-

mosome (i.e., �standard� sex determining system

in house flies).

In summary, we have cloned a nAChR subunit

gene, Mda2, cDNA, and analyzed its gene expres-

sion and genomic organization. The Mda2 was

abundantly expressed in house fly heads and tho-

races and was linked to autosome 2. Given the

importance of neonicotinoid insecticides (Jeschke

and Nauen, 2004), and the potential for resistance

to occur by mutations in the nAChR (Liu et al.,

2005), it is important to determine the sequence

of the nAChR genes as a first step to being able

to examine mutations that might give rise to in-

secticide resistance. This information is a critical

first step toward the development of sensitive

resistance monitoring techniques. In addition,

cloning of nAChR genes is a vital first step in un-

derstanding the function of these physiologically

important receptors.

Fig. 5. Expression of Mda2 in different body parts of adult

aabys house flies measured by quantitative real-time PCR.

Error bars represent standard error of the means of five

replicates. Different letters with the bars indicate that the

means are significantly different (P < 0.05) in Tukey�s test.

TABLE 2. Linkage Analysis of Mda2 in House Flies

Mda2 allele A

Genotypea homozygotes/totalb

+/ac;ar/ar;bwb/bwb;ye/ye;snp/snp 3/3

ac/ac;+/ar;bwb/bwb;ye/ye;snp/snp 0/10

ac/ac;ar/ar;+/bwb;ye/ye;snp/snp 3/3

ac/ac;ar/ar;bwb/bwb;+/ye;snp/snp 2/3

ac/ac;ar/ar;bwb/bwb;ye/ye;+/snp 3/3

aThe recessive markers ac, ar, bwb, ye and snp are on autosomes 1, 2, 3, 4 and 5,

respectively.bThe Mda2 allele A was found in aabys, but not in the OCR strain.

40 Gao et al.


ACKNOWLEDGMENTS

The authors thank C. Gilbert for valuable dis-

cussions, C. A. Leichter for technical assistance and

S. Kasai and F.W. Plapp, Jr., for providing house

fly strains. This work was supported by Elanco Ani-

mal Health and the Daljit S. and Elaine Sarkaria

Professorship. Partial support for J.M.D. was pro-

vided by the Cornell Presidential Research Schol-

ars Program.

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