The Acetylcholinesterase Gene Ace: a Diagnostic Marker for the Pipiens and Quinquefasciatus Forms of the Culex Pipiens Complex

Journal oJ'the American Mosquito Control Association, 14(4):39O_396, l99g Copyright O 1998 by the American Mosquito Control Association, Inc.

THE ACETYLCHOLINESTERASE GENE ACE: A DIAGNOSTIC MARKER FOR THE PIPIENS AND QUINQUEFASCIATUS FORMS OF THE CULEX PIPIENS COMPLEX

DENIS BOURGUET'2 DINA FONSECA,S GwENAEI- vouRcn,, MARIE-pIERRE DUBOIS,I FABRICE CHANDRE,4 CARLO SEVERINIs AND MICHEL RAYMONDI,6

ABSTRACT' The taxonomy of the Culex pipiens complex remains a controversial issue in mosquito sysrem- atics. Based on morphologic characters, 2 allopatric taxa are recognized, namely pipiens (including "molestus") Cx. the form in temperate areas and Cx. quinquefasciatus in tropical areas. Here we report on variability at the nucleotide level of an acetylcholinesterase gene in several strains and natural populations of this species complex. Few polymorphisms were found in coding regions within a subspecies but many polymorphismi were observed between subspecies in noncoding regions. We describe a method based on a restriction enzyme polymorphism in polymerase chain reaction-amplified DNA, in which the presence or absence of one restriction site diicrim- inates Cx. pipiens, Cx. quinquefasciatus, and their hybrids. This technique reliably discriminates mosquitoes from more than 30 worldwide strains or populations. Polymerase chain reaction amplification of specific alleles may also be a useful tool for characterizing specific alleles of each sibling taxon.

KEY WORDS Acetylcholinesterase gene, Culex pipiens complex, diagnostic markeq sibling species, Cule-r torrentium, Culex pipiens "molestus"

INTRODUCTION feed on mammals (mammophily). In contrast, females from epigeous The mosquito Culex pipiens represents a species habitats require a blood meal produce complex that is incompletely understood (see Har- to their first batch of eggs (anautogeny), bach et al. [1985] for a review). Based on morpho- are unable to mate in confined spaces, such as in logic characters, 3 types have thus far been de- laboratory conditions (eurygamy), hibernate during scribed: Culex quinquefasciatus Say (Sirivanakarn the winter (heterodynamy), and have a propensity "molestus" and White 1978), Culex pipiens For- to feed on birds (ornithophily). The same associa- skil (Harbach et al. 1984), and Culex pipiens Lin- tion between physiologic traits and habitat types is naeus (Harbach et al. 1985). The last 2 types are observed in northern Europe and in North Ameri- sympatric and are considered by some authors to can and Australian regions with cold winters (Rou- be ecotypes of the same form (Roubaud 1933; Mat- baud 1933, Marshall and Stanley 1937, Spielman tingly 1951; Pasteur 19771,Barr l98l; Chevillon et 1964, Miles 1976). "molesras") al. 1995a,1998; Vinogradova et al. 1996; Eritja, Culex pipiens (including Cx. p. is 1998), as they are mainly distinguished by ecolog- largely a temperate form, whereas Cx. quinquefas- "mo- ical and physiologic characteristics. Culex p. ciatus is cosmotropical (Mattingly et al. 1951, Barr lestus" breeds in underground urban habitats (hy- 1957). Cx. quinquefascialus is homodynamous, pogeous habitats such as cellars, sanitary spaces stenogamous, and anautogenous. Extensive areas of under buildings, and septic tanks), and Cx. pipiens overlap and hybridization exist in the Middle and breeds in rural open-air habitats (epigeous habitats Far East, North and South America, Australia, and such as brooks, rivers, swamps, ditches, or nny ar- Africa (Barr 1982; Urbanelli et al. 1995, 1997). The tificial open-air collection of water). Females from main morphologic differences between Cx. pipiens hypogeous habitats do not require a blood meal to and Cx. quinquefasciatus are found in the male produce their first batch of eggs (autogeny), are genitalia, and can be quantified using the DV/D ra- able to mate in confined spaces (stenogamy), do not tio (Sundararaman 1949), where DV is the distance hibernate (homodynamy), and have a tendency to from the tip of the ventral arm of the phallosome to its intersection with the dorsal arm and D is the I Institut des Sciences de I'Evolution (UMR 5554), La- distance between the tips of the dorsal arms of the boratoire Gdn6tique et Environnement, Universitd Mont- phallosome. Values of DV/D below 0.2 characteize pellier II, Place Eugdne Bataillon, 34 090 Montpellier, France. Cx. pipiens, whereas values above O.4 characteize 2 Present address: Station de Recherche de Lutte Bio- Cx. quinquefasciatus. Although this ratio has prov- logique, INRA La Minidre, 78285 Guyancourt, France. en to be reliable outside hybrid zones by several 3 Molecular Genetics Laboratory-DZR, National Zoo- authors (Mattingly et al. 1951, Ban 1957), its use logical Park, Smithsonian Institution, Washington, DC, is restricted to adult males. More recently, bio- 20008. chemical and molecular techniques have been used 4 ORSTOM, Laboratoire de Lutte contre les Insectes to find diagnostic markers (Miller et al. 1996, Sev- Nuisibles, BP 5045, 34032 Montpellier, France. 5 Laboratorio di Parassitologia, Istituto Superiore di erini et al. 1996, Crabtree et al. 1997). Recently, Sanitd, Viale Regina Elena, 299, OOl6l Rome, Italy. part of an acetylcholinesterase gene, referred to as 6 To whom correspondence should be addressed. Ace, was cloned for a Cx. pipiens strain (Malcolm

390 Deceranen1998 DIAGNosTIc Mnnren tN C. prplalrs Cotr,tplex 391

et al. 1998), thus offering a new opportunity to the PCR primers. For the DC3, Hilo, McCandless, compare Cx. pipiens and Cx. quinquefasciatus at and Macapi populations, the PCR conditions were the genomic level. Here we report partial sequences identical to those described above but reagents of the Ace locus for different collections of the Cx. from ABVPerkin Elmer (Norwalk, CT) and an MJ pipiens complex. Variation in this region clearly ResearchPeltier thermocycler (MJ Research,Inc., discriminates Cx. pipiens from Cx. quinquefascia- Waterton, MA) were used instead. The PCR prod- /zs. Based on these sequences we propose and test ucts were purifled with a QlAquick PCR purifica- a restriction enzyme pattern as a diagnostic marker tion kit (Qiagen, Valencia, CA). One microliter of for the 2 subspecies. clean DNA was cycle sequencedusing AmpliTaq DNA FS polymerase and dye-labeled terminators (PE Biosystems, Foster City, CA), and was exam- MATERIALS AND METHODS ined on an automated sequencer(ABI/Perkin El- Mosquitoes: Origins and references of the strains mer). and populations used in this study are given in Ta- Restriction fragment length polymorphism ble 1. Mosquitoes from populations or strains close (RFLP) analysis: At least 3 mosquitoesfrom each to putative hybrid zones (Mattingly et al. 1951) strain or population were analyzed except for Fort such as BED (South Africa), Killcare (Australia), Knox and Simpson where RFLP analyseswere per- DC3 (Washington DC, USA), and BEIJING (Chi- formed on genomic DNA of up to 10Omosquitoes. na) were classified as Cx. pipiens or Cr. quinque- Single mosquito genomic DNAs were obtained fol- fasciatus by means of DV/D ratios of male genitalia lowing Qiao and Raymond (1995). The 700-bp (Ban 1957). Females of the strain S-LAB (Cx. fragment of the Ace gene was amplified as de- quinquefasciafus) were crossed with males of 2 dif- scribed above. Aliquots of l0 pl of each amplifi- ferent Cx. pipiens strains from southern France to cation were digested with the ScaI restriction en- obtain hybrid individuals that were referred to as zyme and loaded onto a 1.5-2Vo(w/v) agarosegel MSE-FI and RSV respectively (Table 1). Mosqui- with tris borate EDTA (TBE) buffer. toes from 2 populations of Culex torrentium Mar- tini (see Thble 1) were also used for comparison. Polymerase chnin reaction (PCR) amplification RESULTS AND DISCUSSION and sequencing.' For the MSE, BRUGES A, Praias, Ace polymorphism S-LAB, SUPERCAR, MRES, and BEUING strains, genomic DNA extraction of up to l0O mosquitoes At least 363 nucleotide sites (44 in exon 2, 158 was perfonned as described by Raymond et al. in intron 2, arrd 161 in exon 3) have been se- (1989). The DNA from the DC3. Hilo. and Mc- quenced at the Ace locus for several strains and Candless strains was extracted from individual populations from various geographic areas (China, mosquitoes using a standard phenol-chloroform Hawaii, Brazil, Ivory Coast, Cuba, and California protocol (Sambrook et al. 1989). A 7Oo-base pair for Cx. quinquefasciatusartd France, Belgium, Por- (bp) fragment (which encompassed part of exon 2, tugal, and Washington,DC, for Cx. pipiens). Yari- intron 2, and part of exon 3, see Fig. 1 and Malcolm able nucleotidesare shown in Fig. 2. Variable sites et al. [1998]) of the Ace gene was amplified using are mainly located in intron 2 and substitutions in the oligonucleotide primers F 1457 (5'-GAGGA- the exons did not change the inferred amino-acid GATGTGGAATCCCAA-3') and B 1246 (5'- sequence.This indicates that Ace is probably not a TGGAGCCTCCTCTTCACGGC-3') (Eurogentec, pseudogene,although its exact function remains Seraing, Belgium). Amplifications were performed unknown (Malcolm et al. 1998).The polymorphism in a 50-pl volume containing 75 mM Tfis-HCl (pH zrmongstrains of the same subspeciesis low and 9.O), 20 mM (NH4),SO', O.l7o (w/v) Tlveen 20, Taq er'rorsmay not be excluded. In contrast, we 1.25 mM MgClr, 250 pM of each deoxynucleoside found many differences (37 variable sites out of triphosphate (dNTP), IOO ng of each primer, 10- 7lO sequenced)between the Ace sequencesof Cx. 100 ng of DNA, and 2.5 units of Taq polymerase pipiens and those of Cx. quinquefasciatus.The Ace (Eurogentec). The tubes were then quickly trans- gene of the Cx. pipiens complex is characterizedby ferred to the thermal cycler (Thermocycler Croco- the presenceof 10 introns (Malcolm et al. 1998). dile II, Appligene, Illkirch, France). After 5 min at With the exception of intron 4, these introns are 93oC, reactions were cycled 35 times through the very large, resembling more the structure of the following temperature profile: 93oC for 1 min, 52oC Drosophila melanogasterAce Meigen gene (Four- for 1 min, and 72"C for 90 sec. The tubes were nier et al. 1989) than that of Anopheles stephensi finally incubated at 72"C for 10 min. One hundred (Malcolm and Hall 1990). microliters of PCR products of MSE, BRUGES A, Praias, S-LAB, SUPERCAR, MRES, and BEUING A diagnostic marker were purified (Geneclean II Kit, Bio 101 Inc., Vista, CA) and resuspended in 2O pl HrO. The purified A ScaI restriction site that discriminates Cx. pi- PCR products were then sequenced following the piens from Cx. quinquefasciatusalleles was found procedure described by Rousset et al. (1992) with in intron 2 (Fig. 3). The 700-bp amplified Ace frag- 392 JounulL oF THE AMERTCIN Mosquno CoNTRoL AssocrATroN Vor. 14,No.4

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Fig. 1. Mapof theAce geneintheCulexpipiens complex,indicatingthelocationof exons(boxesE2toE5)and introns (lines 12 to 14) already known, and the amplified fragment used in this study (see Malcolm et al. 1998 for a detailed nucleotide sequence).

ment of Cx. quinquefasciatuspossesses 2 ScaI re- weight and the ^lcal restriction profile did not restriction sites but only one is shared with the Ace semble that of Cx. pipiens or that of Cx. quinque- sequenceof Cx. pipiens (Fig. 3). Thus, the presence fasciatus. or absenceof this restriction site may be used to A PCR assayfor discriminatingthe2 sibling taxa distinguish between the 2 subspecies.This tech- has also been developedby Crabtree et al. (1997). nique has been used on single mosquitoesfrom the By using subtractive hybridization, they isolated a worldwide populations and strainslisted in Table l. DNA fragment containing a sequencespecific to The restriction profile patterns found for Cx. pi- Cx. pipiens. They used this sequenceto design PCR piens, Cx. quinquefasciatus, ard their hybrids are primers that amplifled a specific product from Cx. shown in Fig. 4. All mosquitoes possessan iden- pipiens but not from Cx. quinquefasciafusgenomic tical ScaI site, generating 2 fragments of 470 and DNA. Although use of these primers was one of 230 bp. The extra ScaI restriction site of Cx. quin- the first molecular tools for examining the Cx. pi- quefasciatusalleles cuts the 470-bp fragment into piens complex at the taxonomic level, the method 2 fragments (350 + l2O bp). Culex pipienr mos- does not provide a perfect diagnostic marker. First, quitoes are characterizedby the presenceof 2 ftag- the presence or absenceof PCR product amplifi- ments (470 and 23O bp), whereas Cx. quinquefas- cation segregatesas a dominant marker so that Cx. ciatus has 3 fragments (35O,23O,and 120 bp). Hy- pipiens and Cx. pipiens-Cx. quinquefasciafi.rs hybrid mosquitoes display the 4 predicted bands (470, brids cannot be differentiated. Second, identiflca- 35O,23O,and l2O bp). tion of Cx. quinquefasciatus is based on the ab- "molestus" Culex pipiens and C.r. p. ecotypes sence of amplified product and therefore cannot share a similar ScaI restriction profile, supporting distinquish the presence of Cx. quinquefasciatus the hypothesis that significant gene flow occurs be- DNA from the absenceof adequate template DNA. tween them (Chevillon et al. 1998). Genomic DNA Our assay uses a codominant marker that allows from C-r. torrentium, a species known to be closely identification of each taxon and their hybrids based related to members of the Cx. pipiens complex on distinct restriction profiles. (Miller et al. 1996), was used to determine whether The technique of PCR amplification of specific the presenceof the 2nd Ace ScaI site is a derived alleles first describedby Sommer et al. (1992) has or an ancestral character. The amplified Ace frag- had wide applicability for the determination of ment from Cx. torrentium had a lower molecular point mutations involved in insecticide resistance

EXOnZ Intron2 Exon3

01 1 1 L 1 1 1 1 1 1 2 2222 22222 2 222222222 333333 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 5 6 6 6 6 67 7 6001 4 4 5 67 8 90 0IIr2233 455 667 7 7 I I 9001 1 1 rL223 4559925556 67 7 7 7 8 I 9214 58 900 70 14 4 I 40 600070290 60 4 4 02 4 62597 9 4 57 1235 60L7 I 4729 47I 907 1 3 68 2 403 190267 I I''SE CACCCTTTTAGGCAGTAATACGAGTGACCGGA---GCA_--ATT-TCTTTTTC-GC-CCTTGTGTTC DC3 -. . . , .A. . . .ATTC..TTT. . .T...... T. .G. . . .AA.-C- BRUGESA C...... PRAIAS C......

C,p.quinq. Hi 1o -GATTAAGACA-TGT. . GAGGAGAATG. TAGGATTAAATTTG . CTAA. - -- . -TTAGTAGCAAA-C- l4cCandless TTAAGACA-TGT. . GAGGAGAATG. TAAGATTAATTTTG . CTAAG--- . -TTAGTAGCAAA-C- Macap6 GATTAAGACA-TGT..GAGGAGAATG,TAGGATTAAATTTGGCTAAG----CTTAGTA SLAB TTAATACA-TGT..GAGGAGAATG.T SUPERCAR TTAAGACA-TGT. . GAGGAGAATG. T MRES TTAATACA-TGT.GGAGGAGAATG.T BEIJING TTAATACA-TGT-.GAGGAGAATGTT

Fig.2. Variable nucleotides at the Ace locus in Culex pipiens and, Culex quinquefasciatus. Deletions are indicated by a hyphen. The positions of variable sites in the genomic sequence are given by the number above MSE nucleotides. Position 1 corresponds to the 5' end of exon 2 (position 1 in Fig. 2a in Malcolm et al. 1998).

DECEMBER1998 DrlcNosrrc Mlnxen rN C. prprelr's Colaplex 39s

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