Enhancing genome investigations in the mosquito Culex quinquefasciatus via BAC library construction and characterization
- Paul V Hickner†1,
- Becky deBruyn†1,
- Diane D Lovin1,
- Akio Mori1,
- Christopher A Saski2 and
- David W Severson1Email author
© Severson et al; licensee BioMed Central Ltd. 2011
Received: 21 April 2011
Accepted: 13 September 2011
Published: 13 September 2011
Culex quinquefasciatus (Say) is a major species in the Culex pipiens complex and an important vector for several human pathogens including West Nile virus and parasitic filarial nematodes causing lymphatic filariasis. It is common throughout tropical and subtropical regions and is among the most geographically widespread mosquito species. Although the complete genome sequence is now available, additional genomic tools are needed to improve the sequence assembly.
We constructed a bacterial artificial chromosome (BAC) library using the pIndigoBAC536 vector and Hin dIII partially digested DNA isolated from Cx. quinquefasciatus pupae, Johannesburg strain (NDJ). Insert size was estimated by Not I digestion and pulsed-field gel electrophoresis of 82 randomly selected clones. To estimate genome coverage, each 384-well plate was pooled for screening with 29 simple sequence repeat (SSR) and five gene markers. The NDJ library consists of 55,296 clones arrayed in 144 384-well microplates. Fragment insert size ranged from 50 to 190 kb in length (mean = 106 kb). Based on a mean insert size of 106 kb and a genome size of 579 Mbp, the BAC library provides ~10.1-fold coverage of the Cx. quinquefasciatus genome. PCR screening of BAC DNA plate pools for SSR loci from the genetic linkage map and for four genes associated with reproductive diapause in Culex pipiens resulted in a mean of 9.0 positive plate pools per locus.
The NDJ library represents an excellent resource for genome assembly enhancement and characterization in Culex pipiens complex mosquitoes.
Culex quinquefasciatus (Say), the southern house mosquito, is a major vector for a number of important human pathogens including West Nile virus and Wuchereria bancrofti, the primary global etiologic agent for lymphatic filariasis (LF) [1–3]. It is estimated that more than 1.2 billion people are at risk for infection by parasites causing LF, with 120 million people presently infected . Among these are over 40 million people who suffer from chronic morbidity associated with lymphadema and hydrocele . Despite the availability of effective antihelminthics to treat and prevent infections, the damage to the lymphatic system caused by these parasites is largely irreversible. Although efforts to eradicate LF globally using mass drug administration to human populations in endemic areas were initiated in 2000, the success of these efforts will likely also rely on the implementation of effective mosquito vector control strategies . However, vector control efforts can be hindered by the rapid selection for emergence of insecticide resistance . Consequently, the identification of new targets for insecticides as well as the development of novel vector control strategies is expected to play a large role in the successful control and/or eradication of mosquito-borne diseases .
Cx. quinquefasciatus and Cx. pipiens (L.) are the two most common and geographically widespread species in the Cx. pipiens complex, a species complex with nearly worldwide distribution . Cx. quinquefasciatus is common in tropical and subtropical regions while Cx. pipiens, the northern house mosquito, occupies more temperate regions. Both species are abundant in urban areas where they oviposit in stagnant, and often polluted water. They frequently enter homes and feed on humans during the night, hence the common name of house mosquito. The taxonomic status of this complex has been a subject of debate, and these taxa are sometimes placed within a single species, i.e., Cx. pipiens quinquefasciatus or Cx. pipiens pipiens. Introgression between these species is common in the United States where hybrids can be found as far south as Louisiana and as far north as Illinois [10–12], yet in South Africa the populations remain largely distinct [13, 14]. Females are morphologically indistinguishable, while differences in male genitalia have been used to identify species as well as interspecies hybrids [10–12, 15]. Recently, however, PCR assays have been developed to aid in the differentiation of species in this complex [16–19].
Given their medical importance, Cx. pipiens complex mosquitoes have garnered considerable attention by the scientific community during the last 100 years . Nevertheless, the current status of contemporary Cx. pipiens genetics remains considerably behind that of other important mosquito vectors such as Anopheles gambiae and Aedes aegypti. The Cx. quinquefasciatus (Johannesburg strain) genome sequence was recently determined using the whole genome shotgun (wgs) approach, thus providing a valuable resource for advancing genome studies in this species complex . However, the genome assembly remains highly fragmented and few (~40) of the 3171 supercontigs have been assigned to their respective chromosomes .
Bacterial artificial chromosome (BAC) genomic libraries are important resources for the assembly and characterization of complex genomes. They have been utilized for the assembly of numerous genomes including Drosophila melanogaster and An. gambiae[22, 23]. BAC libraries have also been used for the development of genetic markers for non-model organisms [24, 25]. Furthermore, BAC clones can be used for positional cloning to help identify and characterize genomic regions of interest [26, 27], as well as for construction of BAC-based physical map assemblies [28–31]. These are useful for long-range contiguity and anchoring of wgs draft assemblies as well as targeted re-sequencing for high resolution using BAC pools . The objective of this work was to construct a BAC library with comprehensive coverage of the Cx. quinquefasciatus genome, thereby providing a tool to aid in genome assembly, marker development, and gene discovery in Cx. pipiens complex mosquitoes.
BAC library construction
High molecular weight DNA was extracted from pupae from the Johannesburg (JHB) strain. This strain was established using individuals from Johannesburg South Africa, and was the strain used in the Cx. quinquefasciatus genome project . Pupae were gently homogenized in 1X PBS buffer containing 50 mM EDTA pH 8.0 and 0.1% BME and filtered through one layer of miracloth into 50 mL Falcon tubes. Cells were pelleted by centrifugation in a swinging-bucket rotor (Beckman) at 3,200 rpm for 15 minutes at 4°C. Pellets were washed 2 additional times with PBS and gently resuspended in 1 mL of PBS. The nuclei solution was warmed to 45°C in a waterbath, mixed gently with an equal volume of 1.5% low-melt agarose (Seaplaque) and aliquoted into plug molds (BioRad) using large-bore tips. Protein digestion and plug washing was performed exactly as the methods of Luo and Wing (2003) .
Hin dIII partial restriction enzyme digestion of DNA, as well as the preparation of high molecular weight DNA fragments was conducted following the procedure of Luo and Wing (2003) . Preparation of the Hin dIII cloning-ready single copy pIndigoBAC536 vector from the high copy pCUGIBAC1 plasmid was performed according to Luo et al. (2001) . The size selected high molecular fragments were ligated to the vector and transformed into E. coli strain DH10B competent cells (Invitrogen, Carlsbad, CA). White recombinant colonies were selected on LB plates containing chloramphenicol, X-Gal and IPTG, and picked robotically using the Genetix Q-bot (Genetix, UK). Recombinant clones were transferred into individual wells of microtiter plates, grown and then stored at -80°C. The BAC library was also gridded onto 10, 11.25 × 22.25 cm filters in high density, double spots (18,432 clones represented per filter) and 4 × 4 patterns.
To estimate the size of the BAC inserts, DNA from 82 randomly selected clones was prepared according to standard alkaline lysis protocol, digested with Not I, and separated by pulsed-field gel electrophoresis (PFGE) on a 1% agarose gel under the following conditions: 5-15 sec linear ramp time, 6 V/cm, 14°C in 0.5 × TBE buffer for 15 hours and stained with ethidium bromide. Insert sizes of the clones with endogenous Not I sites, evidenced by multiple restriction fragments, were estimated by summing the fragments. Southern blotting was used to confirm that all of the clones were truly Cx. quinquefasciatus and are not significantly contaminated by other types of DNA. One gel used for insert size determination was transferred to a positively charged nylon membrane Hybond N+ (GE Healthcare) following the methods of Chomczynski (1992) . BAC vector (pIndigoBAC536) and total Culex DNA were used as probes and radiolabeled with the DECAprime™ II kit (Ambion, Inc). The probes were mixed and denatured, and hybridization was carried out overnight at 60°C. The membrane was washed with 1× SSC, 0.1% SDS at 60°C twice for one hour each. The membrane was exposed to a phosphor screen (GE Healthcare) overnight and the image recorded by a Typhoon 9400 imager (GE Healthcare).
BAC library screening
Screening of the BAC library was generally performed as described by Jiménez et al. (2004) . Briefly, we first prepared pools of DNA representing all clones within each of the individual 144 384-well microplates. Plates were initially replicated on LB agar plates containing 12.5 μg/ml chloramphenicol and incubated overnight at 37°C. The plates were then flooded with LB broth containing 12.5 μg/ml chloramphenicol, agitated for 4 h at 37°C and the slurries used to prepare 9.5 ml overnight cultures. These individual plate pool cultures were used for large-scale alkaline lysis DNA extractions  and subsequent PCR screening with SSR oligonucleotide primer sets.
SSR primer sequences.
SSR Primer Sequence (F/R)
Product size (bp)a
# Positive plate pools
Primer sequences for genes used in library screening.
VectorBase gene ID
SSR Primer Sequence (F/R)
Product size (bp)a
# Positive plate pools
Well position of marker loci within select positive microplates was determined by DNA-DNA hybridization. Four individual clones were identified by probing with P32-labeled PCR amplicons (C127GAC1, C65AC1, C99TGT1, and FOXO) and thereafter sized with PFGE. Briefly, microplates representing positive pools were replicated to Colony/Plaque screen hybridization membranes (NEN™, Life Science Products) following Jiménez et al. . Hybridizations and radiolabeling of the target clones were conducted following our standard probing procedures . The presence of the marker locus in each of the four clones was confirmed by PCR and UV visualization on 2% agarose gels, as described for plate pool screening.
Results and Discussion
Detailed genetic and genomic studies among the Cx. pipiens complex could provide valuable insights into the molecular genetic mechanisms influencing important traits such as vector competence, insecticide resistance, and reproductive diapause. Despite morphological similarities and their ability to form hybrid populations, species within the complex differ in several life history traits. For example, Cx. quinquefasciatus requires a blood meal prior to laying eggs (anautogenous) and is unable to enter diapause and overwinter in cold climates. Cx. pipiens and Cx. pipiens pallens also are anautogenous but adult females are able to enter reproductive diapause and survive winter in temperate climates, and Cx. pipiens molestus is able to lay eggs without taking a blood meal (autogenous) but does not enter diapause [45–47]. Presently, detailed molecular analyses of these traits are limited by the fragmented genome assembly. Fingerprinting, end-sequencing and physical assembly of the NDJ BAC library would likely facilitate the construction of a more complete genome sequence assembly by serving as a template for genome finishing, including gap-filling, as well as providing resources to enable the assignment of the individual superscaffolds to their respective chromosome position via in situ hybridization. In summary, the NDJ BAC library provides a valuable resource for marker development, positional cloning, and genome sequence assembly enhancement for Cx. quinquefasciatus thus helping to advance genome studies in Cx. pipiens complex mosquitoes.
The NDJ BAC library is available to researchers through the Clemson University Genomics Institute (see Culex pipiens library CPQLBa at http://www.genome.clemson.edu/).
This work was supported by grant RO1-AI079125 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA.
- Turell MJ, O'Guinn ML, Dohm DJ, Jones JW: Vector competence of North American mosquitoes (Diptera: Culicidae) for West Nile virus. J Med Ent. 2001, 38: 130-134. 10.1603/0022-2585-38.2.130.View ArticleGoogle Scholar
- Fonseca DM, Keyghobadi N, Malcolm CA, Mehmet C, Schaffner F, Mogi M, Fleischer RC, Wilkerson RC: Emerging vectors in the Culex pipiens complex. Science. 2004, 303: 1535-1538. 10.1126/science.1094247.PubMedView ArticleGoogle Scholar
- Michalski ML, Erickson SM, Bartholomay LC, Christensen BM: Midgut barrier imparts selective resistance to filarial worm infection in Culex pipiens pipiens. PLoS Negl Trop Dis. 2010, 4: e875-10.1371/journal.pntd.0000875.PubMedPubMed CentralView ArticleGoogle Scholar
- Michael E, Bundy DA, Grenfell BT: Reassessing the global prevalence and distribution of lymphatic filariasis. Parasitology. 1996, 112: 409-428. 10.1017/S0031182000066646.PubMedView ArticleGoogle Scholar
- Addiss DG, Brady MA: Morbidity management in the global programme to eliminate lymphatic filariasis: a review of the scientific literature. Filaria J. 2007, 6: 2-20. 10.1186/1475-2883-6-2.PubMedPubMed CentralView ArticleGoogle Scholar
- Bockarie MJ, Pedersen EM, White GB, Michael E: Role of vector control in the global program to eliminate lymphatic filariasis. Annu Rev Entomol. 2009, 54: 469-487. 10.1146/annurev.ento.54.110807.090626.PubMedView ArticleGoogle Scholar
- Hemingway J, Ranson H: Insecticide resistance in insect vectors of human disease. Annu Rev Entomol. 2000, 45: 371-391. 10.1146/annurev.ento.45.1.371.PubMedView ArticleGoogle Scholar
- Hill CA, Kafatos FC, Stansfield SK, Collins FH: Arthropod-borne diseases: vector control in the genomics era. Nature Rev Microbiol. 2005, 3: 262-268. 10.1038/nrmicro1101.View ArticleGoogle Scholar
- Vinogradova EB: Culex pipiens pipiens mosquitoes: taxonomy, distribution, ecology, physiology, genetics, applied importance and control. 2000, Sofia: PensoftGoogle Scholar
- Barr AR: The distribution of Culex p. pipiens and Culex p. quiquefasciatus in North America. Am J Trop Med Hyg. 1957, 6: 153-165.PubMedGoogle Scholar
- Cheng ML, Hacker CS, Pryor SC, Ferrell RE, Kitto GB: The ecological genetics of the Culex pipiens complex in North America. Recent Developments in The Genetics of Insect Disease Vectors. Edited by: Steiner WWM, Tabachnick WJ, Rai S, Narang KS. 1982, Champaign: Stipes, 581-627.Google Scholar
- Kothera L, Zimmerman EM, Richards CM, Savage HM: Microsatellite characterization of subspecies and their hybrids in Culex pipiens complex (Diptera: Culicidae) mosquitoes along a north-south transect in the central United States. J Med Ent. 2009, 46: 236-248. 10.1603/033.046.0208.View ArticleGoogle Scholar
- Jupp PG: Culex (Culex) pipiens pipiens Linnaeus and Culex (Culex) pipiens quinquefasciatus Say in South Africa: morphological and reproductive evidence in favour of their status as two species. Mosq Systematics. 1978, 10: 461-473.Google Scholar
- Cornel AJ, McAbee RD, Rasgon J, Stanich MA, Scott TW, Coetzee M: Differences in extent of genetic introgression between sympatric Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in California and South Africa. J Med Entomol. 2003, 40: 36-51. 10.1603/0022-2585-40.1.36.PubMedView ArticleGoogle Scholar
- Sundararaman S: Biometrical studies on intergradation in the genitalia of certain populations of Culex pipiens and Culex quinquefasciatus in the United States. Am J Hyg. 1949, 50: 307-314.PubMedGoogle Scholar
- Crabtree MB, Savage HM, Miller BR: Development of a species-diagnostic polymerase chain reaction assay for the identification of Culex vectors of St. Louis Encephalitis virus based on interspecies sequence variation in ribosomal DNA spacers. Am J Trop Med Hyg. 1995, 53: 105-109.PubMedGoogle Scholar
- Bourguet D, Fonseca D, Vourch G, Dubois M-P, Chandre F, Severini C, Raymond M: The acetylcholinesterace gene Ace: a diagnostic marker for the quinquefasciatus and pipiens forms of the Culex pipiens complex. J Am Mosq Control Assoc. 1998, 14: 390-396.PubMedGoogle Scholar
- Aspen S, Savage H: Polymerase chain reaction assay identifies North American members of the Culex pipiens complex based on nucleotide sequence differences in the acetylcholinesterase gene Ace.2. J Am Mosq Cont Assoc. 2003, 19: 323-328.Google Scholar
- Smith JL, Fonseca DM: Rapid assays for identification of members of the Culex (Culex) pipiens complex, their hybrids, and other sibling species (Diptera: Culicidae). Am J Trop Med Hyg. 2004, 70: 339-345.PubMedGoogle Scholar
- Severson DW, Brown SE, Knudson DL: Genetic and physical mapping in mosquitoes: molecular approaches. Annu Rev Entomol. 2001, 46: 183-219. 10.1146/annurev.ento.46.1.183.PubMedView ArticleGoogle Scholar
- Arensburger P, Megy K, Waterhouse RM, Abrudan J, Amedeo P, Antelo B, Bartholomay L, Bidwell S, Caler E, Camara F, Campbell CL, Campbell KS, Casola C, Castro MT, Chandramouliswaran I, Chapman SB, Christley S, Costas J, Eisenstadt E, Feschotte C, Fraser-Liggett C, Guigo R, Haas B, Hammond M, Hansson BS, Hemingway J, Hill SR, Howarth C, Ignell R, Kennedy RC,et al,: Sequencing of Culex quinquefasciatus establishes a platform for mosquito comparative genomics. Science. 2010, 330: 86-88. 10.1126/science.1191864.PubMedPubMed CentralView ArticleGoogle Scholar
- Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, Scherer SE, Li PW, Hoskins RA, Galle RF, George RA, Lewis SE, Richards S, Ashburner M, Henderson SN, Sutton GG, Wortman JR, Yandell MD, Zhang Q, Chen LX, Brandon RC, Rogers YHC, Blazej RG, Champe M, Pfeiffer BD, Wan KH, Doyle C, Baxter EG, Helt G, Nelson CR, Beasley EM, Berman BP, Bolshakov S, Borkova D, Botchan MR, Bouck J, Brokstein P, Brottier P, Burtis KC, Busam DA, Butler H, Cadieu E, Center A, Chandra I, Cherry JM, Cawley S, Dahlke C, Davenport LB, Davies A, de Pablos B, Delcher A, Deng ZM, Mays AD, Dew I, Dietz SM, Dodson K, Doup LE, Downes M, Dugan-Rocha S, Dunkov BC, Dunn P, Durbin KJ, Evangelista CC, Ferraz C, Ferriera S, Fleischmann W, Fosler C, Gabrielian AE, Garg NS, Gelbart WM, Glasser K, Glodek A, Gong FC, Gorrell JH, Gu ZP, Guan P, Harris M, Harris NL, Harvey D, Heiman TJ, Hernandez JR, Houck J, Hostin D, Houston DA, Howland TJ, Wei MH, Ibegwam C, Jalali M, Kalush F, Karpen GH, Ke ZX, Kennison JA, Ketchum KA, Kimmel BE, Kodira CD, Kraft C, Kravitz S, Kulp D, Lai ZW, Lasko P, Lei YD, Levitsky AA, Li JY, Li ZY, Liang Y, Lin XY, Liu XJ, Mattei B, McIntosh TC, McLeod MP, McPherson D, Merkulov G, Milshina NV, Mobarry C, Morris J, Moshrefi A, Mount SM, Moy M, Murphy B, Murphy L, Muzny DM, Nelson DL,et al,: The genome sequence of Drosophila melanogaster. Science. 2000, 287: 2185-2195. 10.1126/science.287.5461.2185.PubMedView ArticleGoogle Scholar
- Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab R, Nusskern DR, Wincker P, Clark AG, Ribeiro JMC, Wides R, Salzberg SL, Loftus B, Yandell M, Majoros WH, Rusch DB, Lai ZW, Kraft CL, Abril JF, Anthouard V, Arensburger P, Atkinson PW, Baden H, de Berardinis V, Baldwin D, Benes V, Biedler J, Blass C, Bolanos R, Boscus D, Barnstead M,et al,: The genome sequence of the malaria mosquito Anopheles gambiae. Science. 2002, 298: 129-149. 10.1126/science.1076181.PubMedView ArticleGoogle Scholar
- Temnykh S, DeClerck G, Lukashova A, Lipovich L, Cartinhour S, McCouch S: Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential. Genome Res. 2001, 11: 1441-1452. 10.1101/gr.184001.PubMedPubMed CentralView ArticleGoogle Scholar
- Ellison CK, Shaw KL: Mining non-model genomic libraries for microsatellites: BAC versus EST libraries and the generation of allelic richness. BMC Genomics. 2010, 11: e428-10.1186/1471-2164-11-428.View ArticleGoogle Scholar
- Perelygin AA, Scherbik SV, Zhulin IB, Stockman BM, Li Y, Brinton MA: Positional cloning of the murine flavivirus resistance gene. Proc Natl Acad Sci USA. 2002, 99: 9322-9327. 10.1073/pnas.142287799.PubMedPubMed CentralView ArticleGoogle Scholar
- Luo M, Yu Y, Kim H-R, Kudrna D, Itoh Y, Agate RJ, Melamed E, Goicoechea JL, Talag J, Mueller C, Wang W, Currie J, Sisneros NB, RA W, Arnold AP: Utilization of a zebra finch BAC library to determine the structure of an avian androgen receptor genomic region. Genomics. 2006, 87: 181-190. 10.1016/j.ygeno.2005.09.005.PubMedView ArticleGoogle Scholar
- Quiniou SM-A, Waldbieser GC, Duke MV: A first generation BAC-based physical map of the channel catfish genome. BMC Genomics. 2007, 8: e40-10.1186/1471-2164-8-40.View ArticleGoogle Scholar
- Palti Y, Luo MC, Hu Y, Genet C, You FM, Vallejo RL, Thorgaard GH, Wheeler PA, Rexroad CE: A first generation BAC-based physical map of the rainbow trout genome. BMC Genomics. 2009, 10: e462-10.1186/1471-2164-10-462.View ArticleGoogle Scholar
- Fang G-C, Blackmon BP, Henry DC, Staton ME, Saski CA, Hodges SA, Tomkins JP, Luo H: Genomic tools development for Aquilegia: construction of a BAC-based physical map. BMC Genomics. 2010, 11: e621-10.1186/1471-2164-11-621.View ArticleGoogle Scholar
- Xia JH, Feng F, Lin G, Wang CM, Yue GH: A first generation BAC-based physical map of the Asian seabass (Lates calcarifer). PLoS One. 2010, 5: e11974-10.1371/journal.pone.0011974.PubMedPubMed CentralView ArticleGoogle Scholar
- Rounsley S, Marri PR, Yu Y, He R, Sisneros N, Goicoechea JL,et al,: De novo next generation sequencing of plant genomes. Rice. 2009, 2: 35-43. 10.1007/s12284-009-9025-z.View ArticleGoogle Scholar
- Luo M, Wing RA: An improved method for plant BAC library construction. Plant Functional Genomics. Edited by: Grotewold E. 2003, Totowa: Humana, 3-19.View ArticleGoogle Scholar
- Luo M, Wang YH, Frisch D, Joobeur T, Wing RA, Dean RA: Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2). Genome. 2001, 44: 154-162.PubMedView ArticleGoogle Scholar
- Chomczynski P: One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal Biochem. 1992, 201: 134-139. 10.1016/0003-2697(92)90185-A.PubMedView ArticleGoogle Scholar
- Jiménez LV, Kang B, deBruyn B, Lovin DD, Severson DW: Characterization of an Aedes aegypti bacterial artificial chromosome (BAC) library and chromosomal assignment of BAC clones for physical mapping quantitative trait loci that influence Plasmodium susceptibility. Insect Mol Biol. 2004, 13: 37-44. 10.1046/j.0962-1075.2004.00456.x.PubMedView ArticleGoogle Scholar
- Birnboim HC, Doly J: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979, 7: 1513-1523. 10.1093/nar/7.6.1513.PubMedPubMed CentralView ArticleGoogle Scholar
- Keyghobadi N, Matrone MA, Ebel GD, Kramer LD, Fonseca DM: Microsatellite loci from the northern house mosquito (Culex pipiens), a principal vector of West Nile virus in North America. Mol Ecol Notes. 2004, 4: 20-22.View ArticleGoogle Scholar
- Smith JL, Keyghobadi N, Matrone MA, Escher RL, Fonseca DM: Cross-species comparison of microsatellite loci in the Culex pipiens complex and beyond. Mol Ecol Notes. 2005, 5: 697-700. 10.1111/j.1471-8286.2005.01034.x.View ArticleGoogle Scholar
- Hickner PV, deBruyn B, Lovin DD, Mori A, Behura SK, Pinger R, Severson DW: Genome-based microsatellite development in the Culex pipiens complex and comparative microsatellite frequency with Aedes aegypti and Anopheles gambiae. PLoS One. 2010, 5: e13062-10.1371/journal.pone.0013062.PubMedPubMed CentralView ArticleGoogle Scholar
- Rozen S, Skaletsky HJ: Primer3 on the WWW for general users and for biologist programmers. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Edited by: Krawetz S, Misener S. 2000, Totowa: Humana, 365-386.Google Scholar
- Sim C, Denlinger DL: Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens. Proc Natl Acad Sci USA. 2008, 105: 6777-6781. 10.1073/pnas.0802067105.PubMedPubMed CentralView ArticleGoogle Scholar
- Sim C, Denlinger DL: A shut-down in expression of an insulin-like peptide, ILP-1, halts ovarian maturation during the overwintering diapause of the mosquito Culex pipiens. Insect Mol Biol. 2009, 18: 325-332. 10.1111/j.1365-2583.2009.00872.x.PubMedView ArticleGoogle Scholar
- Severson DW: RFLP analysis of insect genomes. The Molecular Biology of Insect Disease Vectors: a Methods Manual. Edited by: Crampton JM, Beard CB, Louis C. 1997, London: Chapman and Hall, 309-320.View ArticleGoogle Scholar
- Tekle A: The physiology of hibernation and its role in the geographical distribution of populations of the Culex pipiens complex. Am J Trop Med Hyg. 1960, 9: 321-330.PubMedGoogle Scholar
- Eldridge BF: Environmental control of ovarian development in mosquitoes of the Culex pipiens complex. Science. 1966, 151: 826-828. 10.1126/science.151.3712.826.PubMedView ArticleGoogle Scholar
- Eldridge BF: The effect of temperature and photoperiod on blood-feeding and ovarian development in mosquitoes of the Culex pipiens complex. Am J Trop Med Hyg. 1968, 17: 133-140.PubMedGoogle Scholar
- Mori A, Severson DW, Christensen BM: Comparative linkage maps for the mosquitoes (Culex pipiens and Aedes aegypti) based on common RFLP loci. J Hered. 1999, 90: 160-164. 10.1093/jhered/90.1.160.PubMedView ArticleGoogle Scholar
- Mori A, Romero-Severson J, Severson DW: Genetic basis for reproductive diapause is correlated with life history traits within the Culex pipiens complex. Insect Mol Biol. 2007, 16: 515-524.PubMedGoogle Scholar
- Lawson D, Arensburger P, Atkinson P, Besansky NJ, Bruggner RV, Butler R, Campbell KS, Christophides GK, Christley S, Dialynas E, Emmert D, Hammond M, Hill CA, Kennedy RC, Lobo NF, MacCallum MR, Madey G, Megy K, Redmond S, Russo S, Severson DW, Stinson EO, Topalis P, Zdobnov EM, Birney E, Gelbart WM, Kafatos FC, Louis C, Collins FH: VectorBase: a data resource for invertebrate vector genomics. Nucleic Acids Res. 2009, 37: 583-587. 10.1093/nar/gkn857.View ArticleGoogle Scholar
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