The Journal of Basic & Applied Zoology (2012) 65, 79–82

The Egyptian German Society for Zoology

The Journal of Basic & Applied Zoology

www.egsz.orgwww.sciencedirect.com

Isolation of acetylcholinesterase gene from Egyptian

Anopheles pharoensis (Theobald)

Nahla M. Wassim

Molecular Biology Unit, Zoology Department, Faculty of Science, Suez Branch, Suez Canal University, Suez, Egypt

Received 4 July 2012; accepted 9 August 2012Available online 22 October 2012

E-

Pe

Zo

20

ht

KEYWORDS

Evolution;

ace-2 gene;

Mosquitoes;

Insecticide resistance

mail address: nahlawassim@

er review under responsibilit

ology.

Production an

90-9896 ª 2012 The Egyptia

tp://dx.doi.org/10.1016/j.joba

yahoo.co

y of the

d hostin

n Germa

z.2012.08

Abstract Acetylcholinesterase (ACE) is the target of two major insecticide families, organophos-

phates (OPS) and carbamates. Acetylcholinesterase is a frequent resistance mechanism in insects

and responsible mutations were identified in many dipteran species. The present work amplified,

sequenced and aligned the entire section of intron II and sections of exon 2 and 3 of ace-2 gene

of Anopheles pharoensis (Theobald) to focus on its evolution mechanism. The amplicons are

545 pb in length and the identity is 66% to Anopheles gambiae. Equal numbers of guanine instead

of 18 adenine, 12 cytosine and 11 thymine, respectively. An equal number of while cytosine instead

18 thymine and 13 adenine. Seven thymine instead the same number of adenine was present. The

highest record of point mutations were 18 purines (G–A) and 18 pyrimidines (C–T). The estimated

transitions/transversion ratio was 0.7.ª 2012 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights

reserved.

Introduction

A number of different malarial vectors have been described inEgypt. Among the most important vectors are Anophelespharoensis, Anopheles multicolor and Anopheles sergenti (Kena-

wy et al., 1990; Kenawy 1991). Unfortunately, as a result ofinsecticide overuse, many vector populations have becomeresistant. By the end of 1985 a total of 50 Anopheline species

were recorded as resistant to one or more pesticides, at least11 of the 50 species being important vectors of malariathroughout the world (WHO, 1986). A number of the vectors

m

Egyptian German Society for

g by Elsevier

n Society for Zoology. Production

.001

are resistant to both organochlorine and organophosphorous

insecticides, but some are also resistant to carbamates andpyrethroids. Among these multiresistant vectors is A. pharoen-sis in Egypt. Some vector populations show exophily to an

extent that makes it difficult to control malarial transmissionby means of vector control based on house spraying alone.Much appears to depend on the environmental conditions

and the genetic background of the vector population. This isparticularly evident in the case of A. pharoensis in Egypt andA. arabiensis in Sudan. This led to develop integrated vectorcontrol programmes involving genetic methods (White,1996).

Acetylcolinesterase terminates synaptic transmissioncholinergic synapses in the central nervous system of insects,by rapid hydrolysis of the neurotransmitter acetylcholine

(Toutant, 1989). Numerous studies have focused on insectAChE because it is the target of organophosphates (Op3)and carbamates, the two major classes of pesticides used for

pest management. Target AChE insensitivity has been de-scribed in many species (Fournier et al., 1993; Fournier and

and hosting by Elsevier B.V. All rights reserved.

Fig. 1 PCR amplification of ace-2 gene of A. pharoensis

mosquitoes. Lane 1–Lane 6 are DNA ladders (100 bp), lane 3

and lane 4 are the amplified fragments of ace-2 gene.

80 N.M. Wassim

Mutero, 1994). To identify the mutation (S) reducing targetsensitivity and thus conferring insecticide resistance, genesencoding AChE (i.e. ace genes) have been sequenced. Evidence

that these genes encoded a functional AChE in cholinergic syn-apses came from the identification, in resistant strains, of pointmutations providing insensitivity toward cholinergic insecti-

cides (Fournier et al., 1993; Fournier and Mutero, 1994; Mute-ro et al., 1994; Martinze-Torres et al., 1998). Recently the acegenes have been cloned and sequenced in Anopheles (A.) gam-

biae, Aedes (A.) aegypti, Culex (C.) pipiens, Drosophila (D.)melanogaster (Bourguet et al., 1998; Martinze-Torres et al.,1998) offering a new opportunity to compare other mosquitospecies at the gene level.

The presence of two nuclear genes that encode AChE wasfirst discovered in C. pipiens (Bourguet et al., 1998; Malcolmet al., 1998) and has been found in other mosquito spp. (Weill

et al., 2002). The ace-1 gene can confer resistance to organo-phosphate insecticides and is therefore subject to selectionpressure. The ace-2 gene is a sex linked gene and its exact func-

tion is unknown (Weill et al., 2002). The aim of this work is topresent a simple molecular test to detect mutations in the ace-2gene which encoded AChE 2 and study the molecular evolu-

tion of this gene in A. pharoensismosquitoes. Intron II and sec-tions of exon 2 and exon 3 of ace-2 gene of A. pharoensis wereselected and compared to the ace-2 gene in A. gambiae.

Materials and methods

Mosquitoes

Mosquito larvae of A. pharoensis have been obtained from theEgyptian Medical Insect Institute, Dokki, Giza, Egypt and

reared under the insectary conditions (temp. 27 �C and 12 hlight–dark) till adult stage and kept at �80 �C until processingfor DNA extraction.

Extraction of genomic DNA

Genomic DNA was extracted from frozen individual mosquito

using Wizard Genomic DNA purification kit (Promega) aspreviously described (Wassim, 2005).

PCR

PCR was performed using the thermo cycler PCR master Mix(Master cycler Gradient 2xT5A- Promega). PCR reaction mix(12.5 ll) was mixed with 20 pmol primers (1 ll each). To am-

plify intron II and sections of exon 2 and exon 3 of ace-2 geneof A. pharoensis, two primers were used, the Foreword primerwas F 1457 (5-GAGGAGATGTGTGGAATCCCAA-3) and

the Reverse primer was B 1246 (50-TGGAGCCTCCTCTT-CACGGC-30). 1 ll of (50:100 lg) of genomic DNA wasextracted genomic DNA, the mix was made upto 25 ll as thetotal volume with deionized freenases water. The PCR reactionwas heated to 93 �C for 5 min and then 35 cycles (1 min at93 �C, 1 min at 52 �C and 90 s at 72 �C and finally 10 min at72 �C and park at 4 �C. The amplified DNA was loaded on

to an agarose gel (2%) with 100 pb loading marker (Promega),stained with ethedium bromide solution 10 mg/1 ml (Promega)and visualized on a UV Transilluminator (Ultra. Lum).

Sequencing analysis

For the sequence analysis, a partial sequence of the ace-2 genewas amplified using F1457and B 1246 s primers, electrophore-

sed, using PCR and sequenced. The gene sequence was alignedwith the Blastn Program Version 1.8.

Results

Sequence analysis of the ace-2 gene

The examined primers were sufficiently specific to amplify theentire section of intron II and sections of exon 2 and exon 3 of

ace-2 gene of A. pharoensis. The amplified fragment rangedapproximately between 500 and 550 pb (Fig. 1). Direct se-quence of this segment showed that the real length of intronII is 242 pb (Fig. 2). While the other parts of segments are

the flanking boundaries of exon 2 which is 167 pb and of exon3 which measures132 pb for ace-2 gene that have beenannealed by the two primers (Fig. 3).

To identify gene encoding AChE2 in A. pharoensis, theBlastn program was used to search for homologues with A.gambiae raw genomic sequences deposited recently in Public

Data Bases A. gambiae AChE2 encoded by ace-2 gene. At least161 nucleotides in the intron II of A. pharoensis are similarwith that in ace-2 gene of A. gambiae (accession number gn/

ti 63228534) and the similarity was 66%. Sequence analysis(Fig. 3) of the ace-2 gene has revealed a few of the point muta-tions. Deletion/insertion events are two. A minimal mis-matches estimate is 20% (83 pb in 405 pb compared). The

gene sequences consisted of equal number of guanine instead18 adenine, 12 cytosine and 11 thymine. As well an equal num-ber of cytosine instead 18 thymine and 13 adenine and seven

thymine instead the some number of adenine was present.The highest record of point mutations were 18 purines(G–A) and 18 pyrimidines (C–T). The transition/transversion

ratio was estimated to be 0.7. An estimate of the time evolu-tionary divergence of mosquitoes A. pharoensis can be derived(4.8 million years) by comparison of nucleotide differenceswithin the ace-2 gene sequence using Sharp and Li (1989).

The sequence of ace-gene of Anopheles mosquitoes are highlydivergent (0.34) between A. pharoensis and A. gambiae.

Fig. 2 Direct sequence analysis of ace-2 gene of A. pharoensis mosquitoes.

Fig. 3 Sequence alignment of the ace-2 gene of A. pharoensis (query) and A. gambiae (subject.)

Isolation of acetylcholinesterase gene from Egyptian Anopheles pharoensis (Theobald) 81

Discussion

The technique of PCR amplification of specific alleles first de-scribed by Sommer et al. (1992) has had wide applicability forthe determination of point mutations involved in insecticideresistance (Martinze–Torres et al., 1998). This technique relies

upon the specific amplification of one allele in preference to theother at a given magnesium concentration within the PCRreaction. The genes encoding the three major targets of con-

ventional insecticides are RDF which encodes a a-amino buty-ric acid receptor subunit; PARA which encodes a voltage-gated sodium channel and ACE, which encodes insect AChE.

Interestingly despite the complexity of the encoded receptorsthat ensues very few amino acid residues are replaced in differ-ent resistant insects; one within RDF, two within PARA andthree or more within ACE (Ffrench Constant et al., 1989).

The recent discovery of the gene coding of the synapticAChE in mosquitoes has led to a paradox in their evolution.Morie et al. (2007) isolated and characterized the complete

genomic DNA sequence for the ace-1 gene in the yellow fevermosquito, Aedes aegypti, which consists of 2721 bp and con-tains a 2109 bp open reading frame that encodes a 702 amino

acid protein. The amino acid sequence is highly conserved withthat of other mosquitoes, including greater than 90% identitywith Culex spp. and about 80% identity with A. gambiae. The

A. gambiae genome contains two ace genes; ace-1, which

encodes the main synaptic AChE (Weill et al., 2002) and

ace-2, which has an unknown function. AChE activity fromace-2 cannot be detected by enzymatic assays. This suggestseither a very low or a very localized enzymatic activity, sug-gesting that it plays a very small role in synaptic AChE. The

ace-2 gene only encodes the main synaptic function in true fliesbut not in other insects (Weill et al., 2002). However, ace-2 isexpressed in all larval instars, the sequence conservation of

ace-2 across the insects may suggest that this gene is beingsubjected to purifying selection, probably related to restrictedsynaptic function and/or other functions.

The present work studied whether there was a faster molec-ular evolution of ace-2 gene in A. pharoensis. Deletion/inser-tion events are common in introns and spacers and are

typically not considered in the calculation of divergence rate.Ignoring the mismatches opposite to the two gaps and the sin-gle pb stretch of differences that could also represent a singlemutational event, generates approximately a mismatch esti-

mate of 34% (83 pb) mismatches in 244 pb compared. Thismismatch estimate combined with the estimate of Sharp andLi (1989) of 16 · 10�9 substitutions per site per year yields a

divergence time of approximately 4.8 million years ago forthe A. pharoensis population. Thus, this time estimate shouldbe viewed only as a rough approximation. Nevertheless, the

A. pharoensis population divergence time estimate is consistentwith the geological record of a major period in Egypt.

82 N.M. Wassim

A relative molecular evolution rate test confirmed that theintensity of purifying select ace-2 gene sequences is constantacross the dipterans irrespective of the presence or absence

of ace-1 gene (Stemfield et al., 1998). These non-synaptic func-tions are probably catalytic, probably operating through thehydrolysis of the same substrate (acetylcholine or ACh) as

the synaptic function (Cousin et al., 2005). This is probablyessential for a possible neofunctionalization toward a synapticfunction, as it is perhaps the case for ace-2 gene in true flies.

Possible non-synaptic functions of the ace-1 gene are yet tobe investigated and if they exist, they may have changed oreven disappeared in true flies (Hurchard et al., 2006). Eitherace-1 gene (e.g. in mosquitoes) or ace-2 gene took over the

function of ace-1 gene during evolution. A phenotypic innova-tion involving ace-2 gene could also be proposed, an adaptivechange concerning ace-2 (e.g. an increase of AChE activity)

could also favor ace-2 in the ace-1/ace-2 activity ratio in syn-apses. There have been several innovations in true flies thatmay have triggered a selected change in ace-2 gene for exam-

ple, adults, have a particularly complex organization of thephotoreceptor synapses characterized by cholinergic presynap-tic platform acting as a sort of signal amplification device

(Meinertzhagen, 1989; Edwards and Palka, 1991; Yasuyamaand Salvaterra, 1999; Buschbeck, 2000; Hurchard et al.,2006). This is interpreted as an adaptation to fast flying, whichrequires particularly efficient and fast receptor–neuron and

neuron–neuron communication. In this situation, even a veryslight increase in activity could be a significant selective advan-tage for ace-2. This changing in the ace-1/ace-2 synaptic activ-

ity ratio could explain the ace-2 takeover of the synapticfunction.

References

Bourguet, D., Fonseca, D., Vourch, G., Dubois, M.-P., Chandre, F.,

Severini, C., Raymond, M., 1998. The acetylcholinesterase gene

ace: a diagnostic marker for the pipiens and quinquefasciatus forms

of the Culex pipiens complex. J. Am. Mosq. Control. Assoc. 14,

390–396.

Buschbeck, E.K., 2000. Neurobiological constraints and fly systemat-

ics: how different types of neural characters can contribute to a

higher level Dipetera. Phylog. Evol. 54, 888–898.

Cousin, X., Strahle, U.T., Chatonnet, A., 2005. Are there non catalytic

function for acetyl cholinesterase? Lessons from mutant animal

models. Bioessays 27, 189–200.

Edwards, J.S., Palka, J., 1991. Insect neural. evolution – a figure or an

opera ? Neuroscience 3, 391–398.

Ffrench Constant, R.H., Pittendrigh, B., Vaughan, Anthony N., 1989.

Why are there so few resistance-associated mutations in insecticide

target genes.? Philos. Trans. Roy. Soc. London Biol. Sci. 353, 1685–

1693.

Fournier, D.T., Mutero, A., 1994. Modification of acetycholinesterase

as a mechanism of resistance to insecticides. Comp. Biochem.

Physiol. C 108, 18.

Fournier, D.T., Mutero, A., Pralavorio, M.T., Bride, J.M., 1993.

Drosophila acetylcholinesterase: mechanism of resistance to orga-

nophosphates. Chem. Biol. Interact. 87, 233–238.

Hurchard, E., Martinez, M., About, H., Douzery, E.T., Phutfalla, G.,

Berthomie, A., Berticat, C., Raymond, M., Waill, M., 2006.

Acetycholinesterase gene within the Diptera, takeover and loss in

true flies. Roy. Soc. B 273, 2595–2604.

Kenawy, M.A., 1991. Development and survival of A. pharoensis and

A. multicolor from Faiyum Egypt. J. Am. Mosq. Control Assoc. 7,

551–555.

Kenawy, M.A., Beier, J.C., Asiago, C.M., el Said, S.E., Roberts, C.R.,

1990. Interpretation of low-level plasmodium infection determined

by ELIZA for anophelines (Diptera:Culicidae) from Egyptian

oases. J. Med. Entomol. 27, 681–685.

Malcolm, C.A., Bourguet, D., Ascolilio, A., Rooker, S.T., Gavery,

C.F., Hall, L.M.C., Pasteur, N.T., Raymond, M., 1998. A sex

linked ace gene not linked to insensitive to acetylcholinesterase

mediate insecticide resistance in Culex pipiens. Insect Mol. Biol. 7,

107–120.

Martinze-Torres, D.F., Chandre, M.S., Williamson, F., Darriet, J.b.,

Berge, A., Devonshere, P., Guillet, N., Pasteur and Pauron, D.,

1998. A molecular dignostic of pyrethroid resistance in the malaria

vector Anopheles gambiae. Insect Mol. Biol. 7, 179–184.

Meinertzhagen, I.A., 1989. Fly photoreceptor synapses their develop-

ment, evolution and plasticity. J. Neurobiol. 20, 276–294.

Morie, A., Lobo, N.F., Bruyn, B.D., Severson, D.w., 2007. Molecular

cloning and characterization of the complete acetylcholinesterase

gene (Ace1) from the mosquito Aedes aegypti with implications for

comparative genome analysis. Insect Biochem. Mol. Biol. 37, 667–

674.

Mutero, A., Pralavorio, M., Bride, J.M., Fournier, D., 1994. Resis-

tance-associated point mutation in insecticide-insensitive acetyl-

cholinesterase. Proc. Natl. Acad. Sci. USA 91, 5922–5926.

Sharp, P.M., Li, W.H., 1989. On the rate of DNA sequence evolution

in Drosophila. J. Mol. Evol. 28, 398–401.

Sommer, S.S., Groszback, A.B., Bottema, C.D.K., 1992. PCR

amplification of specific allele (pasa) in a general method for

rapidly detecting known single base-changes. Biotechniques 12, 82–

87.

Stemfield, M., Ming, G.I., Sela, K., Timberg, R., Poo, M.M., Soreq,

H., 1998. Acetylcholinesterase enhances neurite growth and

synapse development through alternative contributions of its

hydrolytic capacity, core protein and variable c-termini. J. Neuro-

sci. 18, 1240–1249.

Toutant, J.P., 1989. Insect acetylcholinesterase catalytic properties,

tissue distribution and molecular form. Prog. Neurobiol. 32, 423–

446.

Wassim, N.M., 2005. Sequence analysis of the second internal

transcribed spacer region of the ribosomal DNA(ITS2-rDNA)of

Anopheles multicolor (Camboliu)populations from different iso-

lated geographical areas of Egypt. J. Egypt. Ger. Soc. Zool. 161,

161–172.

Weill, M., Fort, P., Berthomieu, A., Dubois, M.P., Pasteur, N.,

Raymond, M., 2002. A novel acetylcholinesterase gene in mosqui-

toes codes for the insecticide, target and is non-homologous to the

ace gene in Drosophila. Proc. Roy. Soc. B 269, 2007–2016.

White, N.J., 1996. The treatment of malaria. N. Engl. J. Med. 335,

800–806.

WHO, 1986. Vector Resistance to Pesticides, Technical Report Series

No. 737.

Yasuyama, K., Salvaterra, P.M., 1999. Localization of choline –

acetyltransferase –expressing neurons in Drosophila nervous sys-

tem. Microsc. Res. Tech. 45, 65–79.