Culex (Culex) Pipiens L

RAPID ASSAY TO IDENTIFY THE TWO GENETIC FORMS OF CULEX (CULEX) PIPIENS L. (DIPTERA: CULICIDAE) AND HYBRID POPULATIONS

CAROLYN M. BAHNCK AND DINA M. FONSECA* Genetics Program, Smithsonian Institution, Washington, DC; Academy of Natural Sciences, Philadelphia, Pennsylvania

Abstract. A previously developed method to identify members of the Culex pipiens complex exploiting polymorphisms in a nuclear intron (acetylcholinesterase [ACE] based-assay) cannot differentiate the two forms of Cx. pipiens: form pipiens and form molestus. Notably, the two forms seem to differ extensively in behavior and physiology and likely have very different epidemiologic importance. Because they are morphologically indistinguishable, molecular methods are critical for the evaluation of their relative importance. Although the two forms of Cx. pipiens have been distinguished using a panel of microsatellite loci, such a protocol is laborious and expensive. We developed a rapid assay based on polymorphisms in the flanking region of a microsatellite locus. Used in conjunction with the ACE-assay, this new assay allows the identification of pure and hybrid populations of the two Cx. pipiens forms as well as those including Cx. quinquefasciatus. We discuss the usefulness of the method as well as limitations to its application.

INTRODUCTION ships between members of the Cx. pipiens complex, we sequenced multiple alleles of several of the microsatellite loci Understanding vector population dynamics is fundamental currently available for the complex.14 During that process, we to the development of disease control strategies. Complexes realized that locus CQ11 was diagnostic for the two forms of of sibling species present unique challenges because of the Cx. pipiens and decided to develop it as a rapid assay. The often-large differences in vectorial capacity between taxa that DNA-based rapid assay designed by Smith and Fonseca15 to are morphologically indistinguishable.1 DNA-based rapid as- identify most of the members of the Cx. pipiens complex, as says have emerged as tools to overcome the challenges of well as other morphologically similar species, does not distin- sibling species identification,2,3 but the Culex pipiens com- guish between the two forms of Cx. pipiens, indicating the plex, which includes important disease vectors,4,5 has proven need for an additional molecular assay. Although there has particularly difficult. This difficulty likely stems from the very been a recent report that several transitions in the mitochon- recent divergence of behaviorally and physiologically distinct drial cytochrome oxidase subunit I allow the identification of sub-groups, possibly associated with domestication. The the two forms,16 that assertion has since been refuted by the nominal species of the complex, Culex (Culex) pipiens L., a authors.17 Here, we describe a rapid assay that can be used to temperate species, has two distinct forms: form pipiens and identify Cx. pipiens f. pipiens and Cx. pipiens f. molestus form molestus.6 Whereas Cx. pipiens f. pipiens diapauses, re- based on indels in the flanking region of a microsatellite locus. quires a blood meal to lay eggs (anautogeny), and is unable to We also expand this assay to recognize the occurrence of mate in confined spaces, Cx. pipiens f. molestus does not populations that include hybrids of the two genetic forms as diapause, is able to lay its first batch of eggs without a blood well as those that include Cx. quinquefasciatus. meal (autogeny), and mates in confined spaces (stenogamy).7 The combination of stenogamy and autogeny seems to allow MATERIALS AND METHODS Cx. pipiens f. molestus to occur in underground areas in urban settings.8 Although conclusive evidence is still lacking, the To prevent the need for cloning, we only identified speci- two forms are thought to have different blood host prefer- mens for sequencing that were homozygous at alleles repre- ences (pipiens biting mainly birds and molestus mainly mam- sentative of the range of allele sizes of CQ11 in Culex pipi- 14 145 mals, especially humans) and therefore very different vecto- ens. We amplified using primer pairs CQ11F2/R3 or 18 rial capacities.9 The two forms have been shown to be geneti- CQ11F/R for Cx. pipiens and Cx. quinquefasciatus, respec- 14 cally isolated in Northern Europe; but there is clear evidence tively, using the conditions in Smith and others. To identify of hybridization in North America.10 Further complicating and discard Taq polymerase errors, two separate independent the epidemiologic landscape in North America, Culex (Culex) amplifications from each individual were sequenced. Cycle quinquefasciatus Say, a tropical and sub-tropical species, hy- sequencing was performed using BigDye Terminator v.3.1 bridizes extensively with the temperate forms (D. M. Fonseca (Applied Biosystems [ABI], Foster City, CA). Sequences and others, unpublished data).10,11 Cx. quinquefasciatus is were analyzed using a capillary automated sequencer (ABI non-diapausing, anautogenous, and stenogamous. Also, as a 3730) and aligned using Sequencher 4.2 (GeneCodes, Ann species, it has no distinct preference for birds or mammals as Arbor, MI). Based on observed diagnostic sequence differ- blood sources and can be a vicious human biter.12,13 Although ences between the two forms, we designed form-specific re- 19 the shape of the male phallosome (genitalia) is diagnostic for verse primers using Primer3. We calculated the number of 20 Cx. pipiens and Cx. quinquefasciatus, the male phallosome is base pair differences between sequences using Arlequin 3.0. indistinguishable between the two forms of Cx. pipiens.6 We excluded the actual microsatellite region contained in With the objective of studying the phylogenetic relation- these sequences from this analysis and express the differences as average percent differences between taxa corrected for intra-taxa variation.20 * Address correspondence to Dina M. Fonseca, Academy of Natural We tested this assay in up to 14 specimens from four popu- Sciences, 1900 Ben Franklin Parkway, Philadelphia, PA 19103. lations of Cx. pipiens f. pipiens, eight populations of Cx. pipi- E-mail: [email protected] ens f. molestus, and four populations of Cx. quinquefasciatus 251 252 BAHNCK AND FONSECA

(Table 1) that were carefully characterized as such by exami- TABLE 2 nation of behavioral and physiologic traits. All the specimens Geographical location and sample sizes (n) of the specimens used to of form molestus were autogenous, bred easily in small cups, compare results of microsatellite analyses and rapid assays and were collected (or their parents were collected) in shel- # Geographical origin n tered areas. Such sheltered areas range from flooded subter- ranean channels in a Menstrie factory and the London sewage Europe 1 BarkingA, England 7 (C. Malcolm, personal communication) to the warm water 2 BarkingB, England 12 surrounding an electrical transformer in a clogged street drain 3 Cogolin, France 8 in Philadelphia in December. The specimens of form pipiens 4 Frejus, France 8 were all collected outdoors, were anautogenous, and did not 5 Madeira Isl, Portugal 13 breed in laboratory cages. We included two locations (Men- 6 MenstrieA, Scotland 8 7 MenstrieB, Scotland 8 strie in Scotland and Barking in London) where “molestus” 8 Nonnenweier, Germany 8 and “pipiens” forms were collected within a few meters of 9 Wiesbaden, Germany 8 each other. Populations of Cx. quinquefasciatus were identi- Americas fied both by genitalia analysis of male specimens as well as by 10 Boston, MA 7 15 11 Chamela, Mexico 9 the acetylcholinesterase (ACE) based rapid assay. 12 Chino, CA 7 To allow the identification of the morphologically very 13 Greenville, SC 9 similar Culex (Culex) torrentium Martini in Europe and Cx. 14 New Orleans, LA 8 quinquefasciatus in North America, we created a composite 15 Philadelphia, PA 13 protocol using the assay we describe de novo here in conjunc- 16 Portland, OR 7 15 17 San Jose, CA 14 tion with the ACE rapid assay. Briefly, the ACE reactions 18 Walkertown, NC 9 contain 1× polymerase chain reaction (PCR) buffer, 250 Total 163 ␮ mol/L of each dNTP, 2 mmol/L MgCl2, 0.15 mg/mL of BSA, Some of the pure populations listed in Table 1 are included above. The symbol # denotes 1 U of AmpliTaq (ABI), 0.1 ␮mol/L of both forward primers the population identifier as it appears in Figure 4. ACEtorr (or ACEquin) and ACEpip, 0.2 ␮mol/L of the re- verse primer B1246s, and 1 ␮L of extracted genomic DNA (∼6 ng). The PCR conditions used were as follows: 94°C for 5 calculated using the program “Structure 2000”21 as in Fonseca minutes and then 35 cycles of 94°C for 30 seconds, 55°C for 30 and others.10 The least-squares regression was performed seconds, and 72°C for 1 minute, concluding with an extension with JMP 3.1.6 (SAS Institute, Cary, NC). To provide a re- of 72°C for 5 minutes. We tested the composite protocol in 26 gression design not biased by an excessive number of pure populations of the Cx. pipiens complex and evaluated the populations, because in such populations the correlation be- ability of this rapid assay to identify hybrid populations. tween the two variables is 1, we removed eight pure popula- We formally assessed the performance of the rapid assay tions of either Cx. pipiens forms or Cx. quinquefasciatus from using a subset of 18 representative populations (Table 2) by the analysis. Their inclusion simply increases the correlation correlating the frequency of “pipiens” alleles in a group of value. specimens to their average probability of Cx. pipiens f. pipiens ancestry based on a full microsatellite analysis as in Smith and Fonseca.15 Probability of Cx pipiens f. pipiens ancestry was RESULTS

We sequenced 10 specimens of Cx. pipiens and7ofCx. TABLE 1 quinquefasciatus. We uncovered extensive variation in the Geographical location and sample size (n) of the pure populations of flanking region of the CQ11 locus and found consistent se- the two forms of C. pipiens as well as of Cx. quinquefasciatus used to test the validity of the rapid assay quence differences between the two Cx. pipiens forms and among all three taxa sequenced (Figure 1). On average, and City Region Country n after correcting for sequence variation within each group, we Culex pipiens f. pipiens determined that Cx. pipiens form pipiens and Cx. pipiens Menstrie* Scotland UK 8 form molestus differed by 14.7% (P < 0.001), a number that Barking* England UK 7 decreases to 2.6% (P < 0.001) when all indels are removed Nonnenweier Baden-Wurttemberg Germany 8 from the analysis. After testing multiple populations of Cx. Altrip Rhenany-Palatinate Germany 8 Culex pipiens f. molestus pipiens f. molestus, we concluded CQ11 had only one allele Menstrie* Scotland UK 8 size, and the microsatellite was composed of just four TG Barking* England UK 12 repeats. In contrast, Cx. pipiens f. pipiens had between 6 and Altenheim Baden-Wurttemberg Germany 8 10 TG repeats (Figure 1). Neuburg Rhenany-Palatinate Germany 7 Exploiting form-specific indels in the left flank of the mic- Wiesbaden Hessen Germany 8 Cernay Alsace France 14 rosatellite region, we developed several alternative diagnostic Philadelphia Pennsylvania USA 13 primers. Ultimately we optimized the protocol for pipCQ11R Amman* Jordan 2 5Ј-CATGTTGAGCTTCGGTGAA-3Ј and molCQ11R 5Ј- Culex quinquefasciatus CCCTCCAGTAAGGTATCAAC-3Ј. The forward primer Archer* Florida USA 7 Ј Ј Chamela* Jalisco Mexico 9 CQ11F2 5 -GATCCTAGCAAGCGAGAAC-3 is the same 18 14 New Orleans* Louisiana USA 8 listed by Fonseca and others as CQ11F. We used a 20-␮L Hawaii* Hawaii USA 10 reaction with a final concentration of 0.1 ␮mol/L of * Populations from which specimens were sequenced. pipCQ11R and 0.15 ␮mol/L of molCQ11R and CQ11F2, 1× ASSAY DIFFERENTIATES THE TWO FORMS OF CULEX PIPIENS 253

FIGURE 1. Polymorphisms occurring in the flanking regions and in the microsatellite locus CQ11 showing the relative position of the diagnostic primers. Each form and species is represented by the inclusive consensus of each allele size sequenced. The size of the alleles in base pairs is listed, and for comparative reasons, we calibrated all alleles to the sizes obtained using primer pair CQ11F2/R3. Position 1 corresponds to the first base pair of the original CQ11 clone, accession no. AF075420. A hyphen indicates a deletion, whereas a period indicates that the sequence is in agreement with Cx. pipiens f. pipiens allele 266. R, A/G; K, G/T; M, A/C; Y, C/T; W, A/T; S, C/G.

␮ PCR buffer, 200 mol/L of each dNTP, 2 mmol/L MgCl2, 0.15 ure 4, with Cx. quinquefasciatus and Cx. pipiens f. molestus mg/mL of bovine serum albumin (BSA),1UofAmpliTaq, having a zero probability of Cx. pipiens f. pipiens ancestry and and 1 ␮L of extracted genomic DNA. Conditions necessary a zero frequency of “pipiens” bands. It is important to note for PCR amplification were as follows: 94°C for 5 minutes and that this analysis could be reproduced using the frequencies of then 40 cycles of 94°C for 30 seconds, 54°C for 30 seconds, the “molestus” or “quinquefasciatus” bands instead. We and 72°C for 40 seconds, concluding with a final 5-minute chose to use frequency of “pipiens” bands because identifi- extension at 72°C. Amplification products were run on a 2% cation of these Cx. pipiens form-specific PCR products in agarose gel. Although the size of the DNA fragments varied hybrid populations does not necessitate the use of the ACE- in Cx. pipiens f. pipiens across the range of microsatellite assay. alleles, they were always obviously smaller than the Cx. pipiens f. molestus DNA fragment. This is caused both by the larger flank of the molestus form (an extra 27 bp) and the fact that the pipCQ11R primer anneals 44 bp downstream from the molCQ11R primer (Figure 1). The size difference allows us to easily distinguish the two forms with a single PCR run on a 2% agarose gel (Figure 2). It is important to point out, however, that the primers are also form specific, so in case of doubt, separate PCRs run with each set of primers will unambiguously identify each form. Although Cx. pipiens f. molestus and Cx. quinquefasciatus differ by 5.3% (1.5% without the indels; Figure 1), we were unable to design a compatible primer that would generate a band in Cx. quinquefasciatus with a diagnostic size. By com- bining our new assay with the ACE-assay, however, we were able to identify both the presence of Cx. quinquefasciatus (or Cx. torrentium in Europe) and American populations containing hybrids of Cx. quinquefasciatus and any of the two Cx. pipiens forms (Figure 3A and B). Specimens of the Cx. pipiens forms and of Cx. quinquefasciatus were identified FIGURE 2. Fragments amplified using pipCQ11R, molCQ11R, unambiguously (Table 1). The probability of identifying hy- and CQ11F2 primers and run on a 2% agarose gel. P, Cx. pipiens f. pipiens (from Nonnenweier, Germany); M, Cx. pipiens f. molestus brid populations was comparable when using the rapid assays (from Altenheim, Germany); MP, f. molestus and f. pipiens hybrid or a full microsatellite analysis (Table 2; Figure 4). Pure popu- (from Greenville, SC); S, size standard (100-bp ladder; New England lations of all three taxa are stacked on either extreme in Fig- Biolabs, Beverly, MA). 254 BAHNCK AND FONSECA

gion of locus CQ11 has already become different in the two Cx. pipiens forms that likely have a very recent shared history (D. M. Fonseca and others, unpublished data). It may also lead some to question the practicality of basing a molecular assay on a marker that we know is variable at the population level.14 However, our assay does not rely on microsatellite length variability; rather, it exploits several large (up to 18 bp) indels in the flanking region of the microsatellite that are specific to either Cx. pipiens f. pipiens or f. molestus. The generally accepted observation that smaller microsatellites experience lower mutation rates22 explains why Cx. pipiens f. molestus is fixed at the CQ11 locus, because replication slip- page, the mechanism believed to generate the majority of microsatellite mutations, can no longer function once a threshold minimum number of repeats is reached. We tested the rapid assay in well-characterized populations FIGURE 3. Amplification products of (A) ACEpip, ACEquin, and of the two forms of Cx. pipiens in northern Europe where B1246s primers run on a 1% agarose gel and (B) pipCQ11R, they do not seem to hybridize and found that it performed molCQ11R, and CQ11F2 primers run on a 2% agarose gel. P, Cx. pipiens f. pipiens; M, Cx. pipiens f. molestus; Q, Cx. quinquefasciatus; well. The unique “pipiens” band was only recovered from −c, negative control; S, size standard (100-bp ladder; New England specimens that matched the behavioral and physiologic char- Biolabs). Multiple letters within a lane denote hybrid ancestry. acteristics of Cx. pipiens f. pipiens, and the converse was true **ACE-assay and CQ11-assay do not agree for this specimen, indi- for the unique “molestus” band. Furthermore, when we cating a recombination event. The sources of specimens used were tested the assay in other populations, we obtained good con- (from left to right): Nonnenweier, Germany; Altenheim, Germany; Archer, FL; Greenville, SC; Walkertown, NC (the last three). cordance between a full microsatellite analysis and the rapid assays. However, because we sampled most extensively from North America and Europe, we are unable to confidently DISCUSSION endorse its use in other areas of the world, particularly in Africa, without further sampling in these locations. We developed a new assay based on variation in the flank- We also found that, in conjunction with the ACE-rapid ing regions of one of the microsatellite loci optimized for Cx. assay, this new protocol provides a fast diagnostic of popula- pipiens, CQ11. Although our current understanding of mic- tions with hybrid specimens that involve one of the two Cx. rosatellite evolution is wanting, it is widely accepted that, in pipiens forms. We emphasize that when hybrids are recog- general, microsatellites mutate at a much higher rate than nized, the full ancestry of the particular individual specimens other nuclear DNA and are usually in hyper-polymorphic requires a full microsatellite analysis. This is so for two rea- 22 regions. This observation may explain why the flanking re- sons: first, the composite assay fails to separate between a hybrid of Cx. quinquefasciatus and Cx. pipiens f. pipiens from a hybrid that also has Cx. pipiens f. molestus ancestry (a three-way hybrid). This is because the pipCQ11R primer am- plifies both in Cx. quinquefasciatus and in Cx. pipiens f. molestus (Figure 1); therefore, a full microsatellite analysis should be used if identifying such hybrids is paramount. Sec- ond, although the rapid assay will identify all first generation (F1) hybrids, backcrossing events result in recombination between loci leading to the independent assortment of the markers within hybrids. As a result, hybrid specimens may lose diagnostic bands. Because recombination occurs ran- domly across independent loci, the likelihood of loosing all traces of a hybrid ancestry decreases as the number of loci examined increases. The decreased power of the rapid assay compared with a full microsatellite analysis, therefore, resides essentially in the lowered number of independent loci it samples. Take, for example, the specimen labeled with two asterisks in Figure 3B; although it generated the diagnostic band for Cx. quinquefasciatus in the ACE locus, it does not have a diagnostic band for that species in the CQ11-assay, indicating recombination. More extensive recombination can lead to hybrid FIGURE 4. Correlation between the frequency of PCR fragments specimens actually lacking both ACE and CQ11 loci that are diagnostic for Cx. pipiens f. pipiens and the average probability of characteristic of one of the parental species or forms. Akin to ancestry from Cx. pipiens f. pipiens based on a panel of eight microsatellite loci across 18 populations from North America and Europe. increasing the number of molecular loci (as in a full micro- The numbers next to each data point (or group of data points) are the satellite analysis), increasing the number of specimens tested population identifiers (#) from Table 2. per population decreases the probability of missing diagnostic ASSAY DIFFERENTIATES THE TWO FORMS OF CULEX PIPIENS 255 markers and leads to correctly identifying a population as scription, variation, and taxonomic status. Proc Entomol Soc containing hybrids. We reiterate, however, that the results Wash 86: 521–542. need to be interpreted at the population and not the indi- 7. Mattingly PF, Roseboom LE, Lloyd E, Knight KL, Laven H, Drummond FH, Christophers SR, Shute PG, 1951. The Culex vidual level. A panel of multivariable microsatellite loci is the pipiens complex. Trans R Entomol Soc Lond 102: 331–382. optimal method for determining the genetic ancestry of indi- 8. Byrne K, Nichols RA, 1999. Culex pipiens in London under- vidual mosquitoes as well as of populations. The rapid assay ground tunnels: differentiation between surface and subterra- we described, however, allows insight into the ancestry of Cx. nean populations. Heredity 82: 7–15. pipiens complex populations without the added expense and 9. Fonseca DM, Keyghobadi N, Malcolm CA, Schaffner F, Mogi M, Fleischer RC, Wilkerson RC, 2004. Reply to outbreak of West time demands associated with a microsatellite analysis. Nile virus in North America. Science 306: 1473–1475. In conclusion, our rapid assay is fast and relatively inex- 10. Fonseca DM, Keyghobadi N, Malcolm CA, Mehmet C, Schaffner pensive and has proven reliable. This approach will be useful F, Mogi M, Fleischer RC, Wilkerson RC, 2004. Emerging vec- in conjunction with ecological studies aiming at ascertaining tors in the Culex pipiens complex. Science 303: 1535–1538. the extent and epidemiologic significance of the multiple taxa 11. Urbanelli S, Silvestrini F, Reisen WK, De Vito E, Bullini L, 1997. Culex pipiens pipiens Cx. in the Cx. pipiens complex, as well as offering insight into the Californian hybrid zone between and p. quinquefasciatus revisited (Diptera:Culicidae). J Med Ento- consequences of their hybridization. The extensive sequence mol 34: 116–127. variation that forms the basis of this new assay also under- 12. Samuel PP, Arunachalam N, Hiriyan J, Thenmozhi V, Gajanana scores the genetic uniqueness of the two forms of Cx. pipiens. A, Satyanarayana K, 2004. Host-feeding pattern of Culex quinquefasciatus Say and Mansonia annulifera (Theobald) (Diptera: Culicidae), the major vectors of filariasis in a rural Received February 9, 2006. Accepted for publication April 4, 2006. area of south India. J Med Entomol 41: 442–446. Acknowledgments: We are deeply grateful to our collaborators for 13. Zinser M, Ramberg F, Willott E, 2004. Culex quinquefasciatus sending us samples. We also thank Kenli Okada and Andrea Widdel (Diptera: Culicidae) as a potential West Nile virus vector in for extensive editorial comments and Tapan Ganguly and the DNA Tucson, Arizona: Blood meal analysis indicates feeding on Sequencing Facility, University of Pennsylvania, for technical assis- both humans and birds. J Insect Sci 4: 20. tance. 14. Smith JL, Keyghobadi N, Matrone MA, Escher R, Fonseca DM, Financial support: This study was supported by NIH Grant 2005. Cross-species comparison of microsatellite loci in the Culex pipiens Mol Ecol Notes 5: R01GM063258 and CDC/NIH Grant U50/CCU220532. complex and beyond. 697–700. 15. Smith JL, Fonseca DM, 2004. Rapid assays for identification of Authors’ addresses: Carolyn Bahnck, Genetics Program, Smithsonian members of the Culex (Culex) pipiens complex, their hybrids, Institution, 3001 Connecticut Ave., NW, Washington, DC 20008- and other sibling species (Diptera: Culicidae). Am J Trop Med 0551, E-mail: [email protected]. Dina M. Fonseca, Academy of Hyg 70: 339–345. Natural Sciences, 1900 Ben Franklin Parkway, Philadelphia, PA 16. Shaikevich EV, Vinogradova EB, 2004. Differentiation between 19103, E-mail: [email protected]. the urban mosquito Culex pipiens pipiens F. molestus and Culex torrentium (Diptera: Culicidae) by the molecular genetic metehods. Parazitologiia 38: 406–412. REFERENCES 17. Vinogradova EB, Shaikevich EV, 2005. Rapid assays for identification of members of the Culex (Culex) pipiens complex, 1. Besansky NJ, 1999. Complexities in the analysis of cryptic taxa their hybrids, and other sibling species (Diptera: Culicidae). within the genus Anopheles. Parassitologia 41: 97–100. Parazitologiia 39: 574–576. 2. Aspen S, Savage HM, 2003. Polymerase chain reaction assay identifies North American members of the Culex pipiens com- 18. Fonseca DM, Atkinson CT, Fleischer RC, 1998. Microsatellite Culex pipiens quinquefasciatus plex based on nucleotide sequence differences in the acetyl- primers for , the vector of avian Mol Ecol 7: cholinesterase gene Ace.2. J Am Mosq Control Assoc 19: 323– malaria in Hawaii. 1617–1619. 328. 19. Rozen S, Skaletsky HJ, 2000. Primer3 on the WWW for general 3. Cockburn AF, 1990. A simple and rapid technique for identifi- users and for biologist programmers. Misener S, Krawetz S, cation of large numbers of individual mosquitoes using DNA eds. Bioinformatics Methods and Protocols: Methods in Mo- hybridization. Arch Insect Biochem Physiol 14: 191–199. lecular Biology. Totowa, NJ: Humana Press, 365–386. 4. Nasci RS, Miller BR, 1996. Culicine mosquitoes and the agents 20. Excoffier L, Laval G, Schneider S, 2005. Arlequin ver. 3.0: An they transmit. Beaty BJ, Marquardt WC, eds. The Biology of integrated software package for population genetics data Diseases Vectors. Niwot: University of Colorado Press, 85–97. analysis. Evol Bioinform Online 1: 47–50. 5. Turell MJ, Sardelis MR, Dohm DJ, O’Guinn ML, 2001. Potential 21. Pritchard JK, Stephens M, Donnelly P, 2000. Inference of popu- North American vectors of West Nile virus. Ann N Y Acad Sci lation structure using multilocus genotype data. Genetics 155: 951: 317–324. 945–959. 6. Harbach RE, Harrison BA, Gad AM, 1984. Culex (Culex) mo- 22. Ellegren H, 2004. Microsatellites: Simple sequences with complex lestus Forskal (Diptera: Culicidae): Neotype designation, de- evolution. Nat Rev Genet 5: 435–445.