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A Set of Broadly Applicable Microsatellite Markers for Analyzing the Structure of Culex pipiens (Diptera: Culicidae) Populations F. E. EDILLO,1 F. TRIPET,2,3 R. D. MCABEE,4 I. M. FOPPA,5 G. C. LANZARO,2 A. J. CORNEL,4 AND A. SPIELMAN Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115

J. Med. Entomol. 44(1): 145Ð149 (2007)

ABSTRACT Microsatellite markers were isolated and developed from Culex pipiens quinquefasciatus Say (Diptera: Culicidae) sampled in Johannesburg, South Africa, to identify those that are broadly useful for analyzing Cx. pipiens complex populations between continents. Suitable loci should be 1) inherited in a codominant Mendelian manner, 2) polymorphic, 3) selectively neutral, 4) randomly associated, 5) without null alleles, and 6) applicable across broad regions and between diverse biotypes. Loci in Cx. p. quinquefasciatus from Johannesburg ranged from two to 17 alleles per locus and expected heterozygosities (He) were 0.02Ð 0.87. Loci in Cx. p. pipiens L. from Johannesburg had Þve to 19 alleles per locus and He values ranging from 0.57 to 0.93, whereas those from George, South Africa, had Þve to 17 alleles per locus and He values ranging from 0.54 to 0.88. Loci in North American mosquitoes were more variable. Cx. p. quinquefasciatus from South Carolina had Þve to 19 alleles per locus and He values ranging from 0.64 to 0.90, whereas Cx. p. pipiens from Massachusetts had six to 28 alleles per locus and with He values ranging from 0.65 to 0.94. All loci were associated randomly. Overall, four of nine of these new loci satisÞed all six criteria for broad utility for analyzing the genetic structure of Cx. pipiens populations. KEY WORDS Culex pipiens complex, Culex p. quinquefasciatus, microsatellite loci

In eastern North America, Culex pipiens pipiens L. and Culex pipiens quinquefasciatus Say (Diptera: Culicidae) maintain transmission of the recently emergent West Nile virus (family Flaviviridae, genus Flavivirus, WNV) as well as a diverse array of other pathogens, worldwide. Deeper understanding of the population structure of these mosquitoes may assist in anticipating the ultimate geographic distribution of WNV infection and in devising effective public health interventions against such pathogens. Microsatellite markers facilitate analyses of the genetic structure of natural populations. To be broadly useful, such markers should be 1) inherited in a codominant Mendelian manner, 2) polymorphic, 3) not subject to selection pressure, 4) randomly associated, 5) without null alleles, and 6) applicable across broad regions. Anoph1

Corresponding author, e-mail: [email protected]. Vector Genetics Laboratory, Department of Entomology and Center for Vectorborne Diseases, University of California at Davis, Davis, CA 95616. 3 Current address: Center for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Staffordshire, ST5 5BG, United Kingdom. 4 Mosquito Control Research Laboratory, Department of Entomology and Center for Vectorborne Diseases, University of California at Davis, Parlier, CA 93648. 5 Department of Epidemiology and Biostatistics, Arnold School of Public Health, University of South Carolina, Columbia, SC 29208. 2

eles gambiae s.l. Giles, Anopheles funestus Giles, and Aedes aegypti L. populations have been the subject of microsatellite studies. A minimum of seven markers has generally been used (Kamau et al. 1998, Braginets et al. 2003, Paupy et al. 2005), although other investigators have opted for 10 or 11 (Lehmann et al. 2003, Cohuet et al. 2005, Tripet et al. 2005), and as many as 17 (Stump et al. 2005) and 21 (Lanzaro et al. 1998, Taylor et al. 2001), depending on the purpose of the study. The Cx. pipiens complex of mosquitoes recently was the subject of a microsatellite analysis that concluded that such mosquitoes in North America are “hybrids” and that they differ epidemiologically from their “nonhybrid” counterparts in Europe (Fonseca et al. 2004). Eight microsatellite markers were used in this study, of which Þve fulÞlled the criteria for broad usefulness, as described above (Fonseca et al. 1998, Keyghobadi et al. 2004). Although 24 microsatellite markers from Cx. pipiens populations are available (Fonseca et al. 1998, Keyghobadi et al. 2004, Smith et al. 2005), only seven fulÞll criteria for analysis of the genetic structure of natural populations. Additional microsatellite loci, therefore, would facilitate critical evaluation of the structure of Cx. pipiens populations. To facilitate genetic analyses of Cx. pipiens populations, we developed an array of microsatellite mark-

0022-2585/07/0145Ð0149$04.00/0 䉷 2007 Entomological Society of America

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ers. Initially, a hybridization enrichment protocol served to isolate microsatellite loci from Cx. p. quinquefasciatus mosquitoes sampled in South Africa. We then developed and characterized primers to amplify these loci based on Cx. pipiens sampled in Africa and North America. The microsatellite markers identiÞed in this study, when added to the existing set (Fonseca et al. 1998, Keyghobadi et al. 2004, Smith et al. 2005), should provide sufÞcient markers for detailed studies on the population structure of these mosquitoes. Materials and Methods Isolation of Microsatellite Loci. To isolate microsatellite loci, genomic DNA was extracted from a pool of 1,800 Cx. p. quinquefasciatus from a laboratory colony originating from 20 gravid females collected from a chicken coop in Johannesburg, South Africa (34⬚ S, 23⬚ E) in 2003. Genomic DNA was sent to Genetic Information Services (GIS), Chatsworth, CA, for enrichment of CA- or GT-, GA-, ATG-, and CAG-microsatellite motifs. GIS digested DNA by using HindIII, ligated to pUC19 plasmid, and the recombinant plasmids were electroporated into Escherichia coli (DH5␣ strain). Clones containing the microsatellite repeats were sequenced to conÞrm their presence. Thirty primer sequences were designed from the sequences ßanking the repeats by using Designer polymerase chain reaction (PCR) version 1.03 (Research Genetics Inc., Huntsville, AL), of which 12 CA- or GT-, eight GA-, three ATG-, and seven CAG-microsatellite primer pairs were synthesized (Integrated DNA Technologies, Inc., Coralville, IA). To ensure repeatability in the ampliÞcation of these microsatellite loci, samples were genotyped both at the Harvard School of Public Health (HSPH) and at the University of California at Davis (UC-D). Optimization trials for each of these loci were conducted in a Þnal volume of 20 ␮l containing 1 ␮l (⬇5 ␮g) of DNA extract, 2 ␮l of 10⫻ PCR buffer, 2 ␮l of 5⫻ Taq enhancer (Eppendorf, Westbury, NY), 0.25 ␮l of 10 mM dNTP (premixed, 10 mM each), 0.12 ␮l of unlabeled forward primer, 0.12 ␮l of ßuorescent-labeled reverse primer, 0.1 ␮l of TaqDNA polymerase (5 U/␮l, Eppendorf, Westbury, NY). PCR was performed in a MJ-PTC-200 (MJ Research, Watertown, MA) or a Mastercycler (Eppendorf, Hamburg, Germany) thermal cycler by using the following program: denaturation step at 94⬚C (5 min), followed by 35 cycles at 94⬚C (40 s), 55⬚C (40 s), 72⬚C (30 s), with an extension step of 72⬚C (15 min). The PCR products were mixed with a GeneScan 400HD (ROX) size standard (PE Applied Biosystems, Foster City, CA) and run on a DNA sequencer (ABI 3100 at UC-D or ABI 3730XL at HSPH). The electropherograms were analyzed using the ABI PRISM GeneScan analysis software, and Genotyper 3.7NT or Genemapper 3.7 DNA fragment analysis software (Applied Biosystems, Foster City, CA) at UC-D or at HSPH, respectively. Characterization of Microsatellite Loci. Microsatellite loci were characterized based on specimens of Cx. p. quinquefasciatus collected from Johannesburg,

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South Africa, and from Columbia, SC (34⬚ N). African Cx. p. pipiens were sampled from Johannesburg and George, located 1,400 km to the south of Johannesburg, and from Cambridge, MA (42.4⬚ N). American mosquitoes were harvested from pans of hay infusion set on the ground, and African mosquitoes as gravid females from which iso-female lines were reared. To determine whether these loci are inherited in a Mendelian codominant manner, we genotyped offspring (F1) samples composed of mosquitoes derived from each of three families of Cx. p. pipiens and Cx. p. quinquefasciatus sampled from North America and from South Africa. To determine whether these loci are broadly useful, we characterized them in numerous families sampled from different locations, each composed of one to two siblings per family. Identity of Mosquitoes. Various criteria were applied to establish the identity of the mosquitoes that were studied. The appearance of the egg-breaker of the Þrst instar served to separate Cx. pipiens from morphologically similar mosquitoes (Dodge 1966). They were identiÞed to subspecies based on the external genitalia of sibling males taken from every family that was subjected to microsatellite analysis. Thus, a DV/D ratio (a convention developed by Sundararaman 1949 to distinguish Cx. p. pipiens from Cx. p. quinquefasciatus) was derived for each family from two African siblings and from six of those from America. A PCR protocol (Crabtree et al. 1995) supplemented these morphological criteria for distinguishing members of Cx. pipiens complex in North American populations. The identity of African mosquitoes was established by the morphology of the derived fourth instar larvae (Cornel et al. 2003) and adult progeny by PCR (Smith and Fonseca 2004). Data Analysis. Goodness-of-Þt for HardyÐWeinberg equilibrium (HWE) by using a probability test for each locus, tests of heterozygote deÞciency, heterozygosities, and pairwise tests for linkage disequilibrium between loci in each population were calculated using GENEPOP 3.4 (Raymond and Rousset 1995). Results Derivation of Microsatellite Loci. First, we derived a series of microsatellite loci that might potentially be useful for describing the population structure of Cx. pipiens mosquitoes. Using the hybridization enrichment protocol, Cx. p. quinquefasciatus sampled from South Africa produced 60 positive clones enriched for microsatellite repeats. These clones were sequenced, and 43 pairs of microsatellite primers were designed, of which 30 pairs were synthesized. Of these Africanderived primers, 15 produced distinct bands on agarose gels. When these African-derived primers were tested with American mosquitoes, nine resulted in ampliÞcation products with similar fragment sizes (Table 1). Mendelian Inheritance of Microsatellite Loci. We tested the Mendelian inheritance of these nine loci by using three families each of Cx. p. quinquefasciatus and of Cx. p. pipiens from Johannesburg. A signiÞcant de-

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Table 1. Primer sequence, repeat motif, allele size range, and GenBank accession number of nine microsatellite loci in Cx. p. quinquefasciatus Locus CxqGT2a CxqCA9a CxqGT108a CxqCA115a CxqCA118a CxqATG9b CxqCAG5b CxqCTG10b CxqCAG101b

Primer sequence (5⬘-3⬘) F: GGTTTGCTGGGAAGAGAGTA R: GTGTCCGTGTGATGAATGAC F: GTGCGGTGTAGTAGTGTGC R: TGTTTTCCAACTGTTTCAGAC F: ACGTGTTTTATAGGCTTCTTTC R: TCTTCCTTAACTTTACCCACTC F: GTCGTCAAACTGCCAATAA R: GCGGAAATAGAACAAACG F: ACCCCGAGCCAACCTTAT R: CCCCCATTTCACACCTGT F: CCACTCAAACTAAAACACCACA R: AATGCCATAACCATCGTCAT F: CACCCCAAATGGGTCAAC R: CGGGATTCATGGGCATAC F: CGTTCTCCAACTGTCATCTTTC R: AACCGAAATCGGAAGGTATTAA F: CAATCAGGGAACCTCAATC R: GGGACTGGGTATTAGGAGAC

Repeat motif

Allele size range (bp)

GenBank accession no.

98Ð228

EF011729

(CA)16

172Ð370

EF011730

(GT)18

100Ð214

EF011731

(CA)12AA(CA)3

157Ð289

EF011732

(CA)12

114Ð282

EF011733

(ATG)7

184Ð262

EF011734

(CAG)9

163Ð307

EF011735

89Ð174

EF011736

174Ð249

EF011737

(GT)12 TT(GT)2

(CTG)4TTG(CTG)4 (CAG)6

The optimal annealing temp is 55⬚C for all primer pairs (F, forward; R, reverse). a Dinucleotide. b Trinucleotide.

parture from the Mendelian pattern was observed at the CxqCA118 locus in two families of Cx. p. quinquefasciatus (␹2 test, P ⬍ 0.001) and at the CxqCAG5 locus in one family of Cx. p. pipiens (␹2 test, P ⬍ 0.001). All other loci were consistent with Mendelian ratios. We also examined the inheritance of all loci in three families of Cx. p. quinquefasciatus from Columbia, SC, and three families of Cx. p. pipiens from Cambridge, MA. Two loci (CxqCA9 and CxqCA115) in one family (␹2 test, P ⬍ 0.001) of Cx. p. quinquefasciatus from South Carolina departed from Mendelian expectation. CxqCAG5 locus in two families (␹2 test, P ⬍ 0.001) and CxqA118 locus in one family (␹2 test, P ⬍ 0.001) of Cx. p. pipiens from Massachusetts deviated from Mendelian proportions. Characteristics of Microsatellite Loci in South Africa. To characterize microsatellite loci in African Cx. p. quinquefasciatus, we calculated gene and genotypic frequencies of two to 17 alleles per locus. Expected heterozygosities ranged widely (Table 2). Although seven loci were highly polymorphic, the CxqCTG10 and CxqCAG101 loci were less so. Based on sequential Bonferroni correction for multiple tests, all but two loci, CxqCA9 and CxqCA115, conformed consistently to HWE. Heterozygote deÞciency suggested selection against particular heterozygotes or the presence of null alleles. Pairwise tests for linkage disequilibrium, incorporating a sequential Bonferroni correction, indicated that all nine loci were randomly associated. Of the nine microsatellite loci that were isolated from African Cx. p. quinquefasciatus mosquitoes, seven seem to be useful for population genetics studies. To characterize the nine loci in African Cx. p. pipiens, we observed Þve to 19 alleles per locus in mosquitoes sampled from George. Expected heterozygosities were uniformly high (Table 2). One locus, CxqCAG5, departed from HWE, with heterozygote deÞciency, and all were randomly associated. Cx. p. pipiens sampled from Johannesburg had Þve to 19

alleles per locus, expected heterozygosities were consistently high (Table 2). SigniÞcant departures from HWE were observed with a deÞcit of heterozygotes at CxqGT2, CxqCA115, and CxqCA118 loci. All loci were randomly associated. Characteristics of Microsatellite Loci in North America. Analyses for general microsatellite applicability were then extended to Cx. p. quinquefasciatus samples from North America. A South Carolina population, containing Þve to 19 alleles per locus, was highly variable (Table 2). None deviated from HWE, and all were in linkage equilibrium. A population of Cx. p. pipiens from Massachusetts was similarly variable (Table 2). Six to 28 alleles were observed per locus. Three loci (CxqCA9, CxqCA118, and CxqCAG5) deviated from HWE, with heterozygote deÞciency. Linkage equilibrium was detected. Discussion This analysis provides nine novel microsatellite loci that may serve as useful markers for analyzing the structure of Cx. pipiens populations. To be broadly useful, however, such loci must be codominant, inherited in a Mendelian manner, polymorphic, selectively neutral, randomly associated, without null alleles, and applicable across broad regions. Of these nine candidate loci, four (CxqGT108, CxqATG9, CxqCTG10, and CxqCAG101) satisfy all of these criteria. These observations prepare the way, ultimately, for studies on the population structure of Cx. pipiens complex. Because they deviate from HWE, the remaining Þve loci should be used with caution. The CxqCAG5 locus deviates similarly among Cx. p. pipiens populations in both continents but does not deviate among those of Cx. p. quinquefasciatus. The CxqCA115 locus deviates among Cx. p. quinquefasciatus populations in Africa and occasionally in South Carolina but not among Cx.

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Table 2. Characterization of nine microsatellite loci in Cx. p. quinquefasciatus and Cx. p. pipiens collected from South Africa and North America Locus, no. of alleles, He(Ho) CxqGT2 No. of alleles He (Ho) CxqCA9 No. of alleles He (Ho) CxqGT108 No. of alleles He (Ho) CxqA115 No. of alleles He (Ho) CxqA118 No. of alleles He (Ho) CxqATG9 No. of alleles He (Ho) CxqCAG5 No. of alleles He (Ho) CxqCTG10 No. of alleles He (Ho) CxqCAG101 No. of alleles He (Ho)

Cx. p. quinquefasciatus

Cx. p. pipiens

Johannesburg 2003 (n ⫽ 30)

Johannesburg 2004 (n ⫽ 48)

Columbia, SC 2004 (n ⫽ 30)

Johannesburg 2005 (n ⫽ 26)

George 2004 (n ⫽ 20)

Cambridge, MA 2004 (n ⫽ 30)

6 0.80 (0.83)

9 0.80 (0.92)

15 0.82 (0.72)

16 0.91 (0.56)

12 0.88 (0.95)

28 0.94 (0.87)

6 0.73 (0.47)

7 0.76 (0.44)

10 0.77 (0.83)

14 0.80 (0.68)

7 0.82 (0.80)

10 0.81 (0.50)

8 0.79 (0.77)

9 0.79 (0.77)

17 0.90 (0.93)

12 0.91 (0.92)

9 0.85 (0.95)

10 0.91 (0.93)

15 0.87 (0.47)

17 0.83 (0.42)

19 0.86 (0.80)

15 0.88 (0.54)

8 0.54 (0.40)

13 0.80 (0.63)

7 0.72 (0.50)

10 0.65 (0.54)

11 0.76 (0.73)

19 0.93 (0.69)

10 0.86 (0.90)

21 0.86 (0.47)

7 0.69 (0.67)

6 0.71 (0.62)

14 0.85 (0.80)

13 0.84 (0.77)

6 0.75 (0.60)

13 0.87 (0.90)

4 0.69 (0.57)

5 0.63 (0.65)

8 0.76 (0.70)

6 0.57 (0.46)

17 0.85 (0.65)

19 0.93 (0.47)

2 0.06 (0.07)

2 0.02 (0.02)

7 0.78 (1.00)

5 0.60 (0.50)

5 0.73 (0.85)

8 0.81 (0.97)

3 0.27 (0.30)

3 0.22 (0.21)

5 0.64 (0.70)

7 0.74 (0.81)

5 0.67 (0.85)

6 0.65 (0.77)

He and Ho indicate expected and observed heterozygosities, respectively.

p. pipiens populations on either continent. The CxqCA9 locus deviates both in Africa and in Massachusetts, but not in South Carolina. The CxqCA118 locus departs from HWE among Cx. p. pipiens samples from Massachusetts and Johannesburg, but is consistent elsewhere. The CxpGTA2 locus deviates only among Cx. p. pipiens from Johannesburg. Although such deviations generally are attributed to differential selection, nonrandom mating produces similar aberrations. Null alleles also create deviations from HWE by producing an apparent deÞciency of heterozygotes (Lanzaro et al. 1998, Lehmann et al. 2003). Apparently similar heterozygosity was present in male (n ⫽ 50) as in female Cx. p. quinquefasciatus (n ⫽ 50) sampled from Johannesburg, suggesting that these loci are autosomal. Because parental specimens of our Þeld-derived mosquitoes were not available, no test of heritability of alleles was conducted. Barriers to gene ßow exist in the Cx. pipiens complex. These mosquitoes exploit sites wherever people disturb the land, from the subarctic to the tropics, and on virtually all continents. Cx. p. pipiens occurs north of latitude 39⬚ N and Cx. p. quinquefasciatus south of 36⬚ N. Intermediates occur between these latitudes (Vinogradova 2000, Spielman 2001). Even in particular sites, autogenous Cx. p. pipiens are isolated from their anautogenous counterparts. Genetic divergence derives from these ecologically distinct adaptations. We now add nine microsatellite markers to those already available in the literature (Fonseca et al. 1998, Keyghobadi et al. 2004, Smith et al. 2005), including

four that can be used for analyzing Cx. pipiens complex populations across broad regions.

Acknowledgments We thank the reviewers for helpful comments on an early version of the manuscript and for A. E. Kiszewski for help with the manuscript. This research was supported by grants RO1AI 52284 to AS and RO1A1 55564 to A.J.C. from the National Institute of Allergy and Infectious Disease, from the University of California Mosquito Research Program to A.J.C., and funds provided by the Centers for Disease Control and Prevention under grant R01AI 44064 from the National Institutes of Health to A.S.

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