Molecular Ecology Notes (2006) 6, 680– 682

doi: 10.1111/j.1471-8286.2006.01303.x

PRIMER NOTE Blackwell Publishing Ltd

Polymorphic microsatellite loci from the West Nile virus vector Culex tarsalis J A S O N L . R A S G O N ,*† M E E R A V E N K A T E S A N ,*† C A T H E R I N E J . W E S T B R O O K *† and M A R Y C L A I R E H A U E R *† *The W. Harry Feinstone Department of Molecular Microbiology and Immunology, and †The Johns Hopkins Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University, Suite E5132 N. Wolfe Street, Baltimore, Maryland 21205, USA

Abstract Since its introduction in 1999, West Nile virus (WNV) has spread across North America. Culex tarsalis is a highly efficient WNV vector species. Many traits such as virus susceptibility, autogeny and host preference vary geographically and temporally in C. tarsalis. Culex tarsalis genomic libraries were developed and were highly enriched for microsatellite inserts (42 –96%). We identified 12 loci that were polymorphic in wild C. tarsalis populations. These microsatellites are the first DNA-based genetic markers developed for C. tarsalis and will be useful for investigating population structure and constructing genetic maps in this mosquito. Keywords: disease vector, microsatellites, mosquito, West Nile virus Received 14 December 2005; revision accepted 9 January 2006

Since 1999, West Nile virus (WNV) has invaded and spread across North America and has been responsible for large epidemics in the western USA (CDC 2004). Culex tarsalis Coquillett (Diptera: Culicidae) is a highly efficient vector species that rapidly becomes infectious after ingesting an infected bloodmeal and transmits the virus at a high rate both orally and vertically (Goddard et al. 2002, 2003). Prior studies have documented spatial variation in the ability for C. tarsalis to transmit WNV both orally and vertically (Goddard et al. 2002, 2003). This variation could be partially explained by genetic differences among C. tarsalis populations, similar to Aedes aegypti and dengue virus (Black et al. 2002). Except for one study investigating allozyme polymorphism among 12 western US populations (Gimnig et al. 1999), little is known about the population structure of C. tarsalis. Although there have been many studies on the ecology and vector potential of this mosquito (e.g. Reisen et al. 1995, 2004), no molecular markers other than allozymes have been developed. We therefore sought to develop polymorphic microsatellite markers suitable for genetic studies in C. tarsalis. Wild adult mosquitoes were collected in California by CDC light traps from Riverside and Los Angeles counties. Correspondence: Jason L. Rasgon, Fax: 1-410-955-0105; E-mail: [email protected]

Colony specimens used for microsatellite-enriched library construction were from the Bakersfield strain (BFS), which has been in continuous culture since 1953 (W. Reisen, personal communication). Library construction followed the methods of Jones et al. (2002). Briefly, genomic DNA from approximately 100 adult males and females was extracted by salt extraction/ethanol precipitation as described by Rasgon & Scott (2003) and partially restricted with a mixture of seven blunt-end cutting enzymes (BsrB1, EcoRV, HaeIII, PvuII, RsaI, ScaI, StuI). Fragments in the size range of 300–750 bp were ligated to oligonucleotide adaptors containing a HindIII restriction site. Magnetic bead capture with 5′-biotinylated capture molecules (CPG) was used to enrich for DNA fragments containing microsatellite repeats. Four libraries were prepared in parallel using capture molecules biotin (AC)12, biotin (AG)12, biotin (CAG)8 and biotin (ATG)8. Captured molecules were amplified and restricted with HindIII to remove the adapters. The resulting fragments were ligated into the HindIII site of pUC19. Recombinant molecules were electroporated into Escherichia coli strain DH5α. From each library, we randomly picked and sequenced 19–27 clones. Plasmid DNA was isolated using QIAprep columns (QIAGEN) and inserts sequenced using primer M13F on an ABI PRISM 3100 Avant Genetic Analyser with BigDye chemistry (Applied Biosystems). In total, out of 98 © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd

© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd

Table 1 List of identified polymorphic Culex tarsalis microsatellite loci. Locus names are followed by GenBank Accession nos. N, number of mosquitoes assayed

Locus

Label

Core repeat

Ta (°C)

Primers

Population

N

CUTA1 DQ296482 CUTA11 DQ296483 CUTB1 DQ296484 CUTC6 DQ296485 CUTC12 DQ296486 CUTC105 DQ296487 CUTD4 DQ296488 CUTD105 DQ296489 CUTD107 DQ296490 CUTD113 DQ296491 CUTD114 DQ296492 CUTD120 DQ296493

6-FAM

(AC)n

60

Los Angeles Co.

25

6-FAM

(AC)n

60

Los Angeles Co.

HEX

(GA)n(AGA)n(GA)n(AG)n

61

HEX

(ATG)n

59

6-FAM

(ATG)n

61

6-FAM

(ATG)nTTGGTGATGCTG(ATG)n

60

HEX

(CAG)n

59

*

(CAG)n

59

6-FAM

(CAG)nCAA(CAG)nCA(ACA)n

64

HEX

(GCA)n

60

*

(GCA)nAGA(GCA)nAGA(GCA)nGAAGCAACA (GCA)nACA(GCA)nACAGCAACA(GCA)n (CAG)n

57

F: 5’-CTCAAACAGCAGAAAGTGACC-3’ R: 5’-ACAAACAATCCATTCCAAGTG-3’ F: 5’-AGCCAGTCAGTCAGTCAGTG-3’ R: 5’-CTCACACCCGATGGTAGAG-3’ F: 5’-GAAAAAAAGGCGCAACATT-3’ R: 5’-GAAGGTGCCAGCCTACTTG-3’ F: 5’-GCGTTTGTCATCTGGTGG-3’ R: 5’-GGGTTCGGAGCAGGAGTA-3’ F: 5’-GTGGAGAACCCGTATTCAAC-3’ R: 5’-TACAATCACGACTCGCACATA-3’ F: 5’-GCCGGTTGTTGTTGTTGTAC-3’ R: 5’-TCCTCGTCAATTTCATCGAC-3’ F: 5’-CGACGAACTCTTATCCGAG-3’ R: 5’-GGAAACCGAAACACATCC-3’ F: 5’-GACCACAGCCAAAAGGTTTAC-3’ R: 5’-TTAGCGAAGGCAGATTTCC-3’ F: 5’-ATGCCGACAGGGAGTTTC-3’ R: 5’-CAAAGGTCTCACGACAGAGC-3’ F: 5’-ATCATACCACTGCCCATAGTC-3’ R: 5’-AACCAGCAGGGACAAGTC-3’ F: 5’-AGGAAGAGTGGTTCGTTTTC-3’ R: 5’-GGGTAAGTTTCAGGGCTATC-3’ F: 5’-TACCCTCGCAAACAAAACAA-3’ R: 5’-GTCGGCTTCCATTCCACTAC-3’

6-FAM

60

No. of alleles

Size range

HO

HE

P

7

116–176

0.400

0.624

0.002

25

15

258–296

0.400

0.940

<0.0001

Los Angeles Co.

25

21

104–146

0.680

0.936

<0.0001

Los Angeles Co.

25

5

216–228

0.240

0.384

0.077

Los Angeles Co.

25

11

184–211

0.680

0.824

0.139

Los Angeles Co.

25

6

212–227

0.680

0.693

0.107

Los Angeles Co.

25

6

208–220

0.348

0.699

<0.0001

Riverside Co.

11

3

220

1.000

*

*

Los Angeles Co.

25

4

184–196

0.333

0.384

0.697

Los Angeles Co.

25

4

167–176

0.600

0.575

1.000

Riverside Co.

11

6

180

0.450

*

Los Angeles Co.

25

4

171–180

0.480

0.744

* 0.079

P R I M E R N O T E 681

682 P R I M E R N O T E clones, 79 contained a microsatellite (77 unique). Fifty-five loci had sufficient flanking sequence for primer design. Primers were designed using primer 3 (http://biotools. umassmed.edu/bioapps/primer3_www.cgi) and amplification optimized using temperature gradient polymerase chain reaction (PCR). PCR was conducted in 25 µL reactions containing 2.5 U Taq polymerase, 2.5 µL ThermoPol buffer (New England Biolabs), 0.5 mm of each dNTP, 1 µm each primer and 1 µL template DNA. Amplicons were amplified using a PTC-200 Peltier thermocycler (Biorad) using a protocol of 95 °C for 5 min, followed by 35 cycles of 95 °C for 1 min, empirically determined annealing temperature (Table 1) for 1 min, 72 °C for 1 min, followed by a 10 min 72 °C extension. Reliable amplification for each primer set was confirmed using 1% agarose gel electrophoresis on wild and BFS individuals. Twenty loci amplified reliably from all individuals tested, and preliminary estimates of allelic variation were determined by denaturing polyacrylamide gel electrophoresis (11% acrylamide, 30% formamide, 5.6 m urea) using specimens from Riverside Co. Gel temperature was held constant at 40 °C by circulating 50% ethylene glycol and amplified fragments separated at 10 V/cm/gel for 4 – 6 h depending on fragment size. Allele sizes were estimated using a 10-bp ladder (Invitrogen). Gels were stained with SybrGreen II (Cambrix) and visualized with ultraviolet light. Four of these loci were monomorphic, and another four appeared to be multicopy and were not investigated further. For the 12 remaining loci, locus CUTD105 and locus CUTD114 were not further characterized. All individuals tested were heterozygous at locus CUTD105 limiting its usefulness as a population genetics marker, although it may still be useful for genetic mapping studies. Locus CUTD114 had an unusually complex core repeat (Table 1). For the remaining 10 loci, the forward primer was 5′ labelled with a HEX or 6-FAM tag and PCR conducted as described. Mosquitoes from Los Angeles Co. (N = 25) were assayed for allelic variation on an ABI-3100 Avant capillary sequencer (Applied Biosystems). Allele sizes were automatically estimated using genescan version 3.7 with an internal ROX-500 size standard (Applied Biosystems). Deviations from Hardy–Weinberg expectation (HWE) and linkage disequilibrium (LD) between loci were calculated with exact tests using genepop software (Raymond & Rousset 1995). Six loci (CUTC6, CUTC12, CUTC105, CUTD107, CUTD113 and CUTD120) were in HWE. The remaining loci showed evidence of heterozygote deficiency, probably due to the presence of null alleles (Table 1). Significant LD was detected between loci CUTA1 and CUTD113 and

between CUTD113 and CUTC105. These observations should be taken into account when using these markers for genetic analysis. The microsatellite loci presented here are the first molecular DNA-based genetic markers developed for C. tarsalis. We anticipate that these markers will be useful for conducting population genetic and mapping studies (Black et al. 2002) in C. tarsalis and possibly other Culex species. Ultimately, these and future markers will make it possible to investigate relationships between population structure and phenotypic variation in such traits as pathogen transmission, autogeny and feeding behaviour in this important vector species.

Acknowledgements We thank W. Reisen, H. Lothrop and L. Kramer for providing mosquito specimens, M. Petridis for laboratory assistance and M. Todd for helpful advice. Funding was provided by the Johns Hopkins Malaria Research Institute.

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© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd