Molecular Ecology Notes (2005) 5, 697–700 doi: 10.1111/j.1471-8286.2005.01034.x © 2005 Blackwell Publishing Ltd Blackwell Publishing, Ltd. TECHNICAL NOTE Cross-species comparison of microsatellite loci in the Culex pipiens complex and beyond JULIE L. SMITH,*†† NUSHA KEYGHOBADI,*§†† MICHAEL A. MATRONE,†¶ RICHARD L. ESCHER‡ and DINA M. FONSECA* * Genetics Program, 3100 Connecticut Ave. NW, Smithsonian Institution, Washington, DC, 20008, USA, † Academy of Natural Sciences, 1900 Benjamin Franklin Pkwy, Philadelphia, PA, 19103, USA, ‡ Florida Medical Entomology Laboratory, 200 9th Street S.E., Vero Beach, FL 32962, USA Abstract In the past, we have developed microsatellite loci from the two most common members of the Culex pipiens complex, Culex quinquefasciatus and Culex pipiens. Here we describe seven additional loci and present an extensive survey of a panel of 20 loci across most of the species and subspecies in the complex as well as in morphologically related species. Because we observed a high degree of polymorphism in the flanking regions, we designed new primers and surveyed multiple populations. We present alternate primers and discuss the cross-species usefulness of these Culex microsatellite loci in a phylogenetic context. Keywords: cross-species amplification, Culex pipiens complex, flanking region polymorphism, microsatellites, population genetics Received 9 February 2005; revision accepted 1 April 2005 Mosquitoes in the Culex pipiens complex vector West Nile virus and St. Louis encephalitis in the eastern USA (Tsai & Mitchell 1989; Turell et al. 2001) and periodic lymphatic filariasis ( Wuchereria bancrofti ), avian malaria, and other encephalitides across the world (Nasci & Miller 1996). The C. pipiens complex includes two worldwide species, Culex pipiens and Culex quinquefasciatus , a north Asian subspecies, Culex pipiens pallens and two species restricted to Australia , Culex australicus and Culex globocoxitus . Furthermore, Culex torrentium in northern Europe , Culex pervigilans in New Zealand , Culex vagans in Asia and Culex restuans and Culex salinarius in North America, are commonly confused with mosquitoes in the C. pipiens complex. Population genetic studies have thus far only been con- ducted on C. pipiens and Cx. quinquefasciatus, the two species from which microsatellite loci have been developed (Fonseca et al . 1998; Keyghobadi et al . 2004). These have yielded unexpected results regarding the origin and genetic makeup of introduced populations (Fonseca et al . 2000; Fonseca et al . 2004). Rapid molecular assays have been developed that distinguish these various taxa (Crabtree et al . 1995; Smith & Fonseca 2004) and further microsatellite-based analyses of population structure on other members of the complex and related species may elucidate the evolution, vectorial capacity and strategies of management of these mosquito species. Here we introduce seven novel microsat- ellites developed from Cx. quinquefasciatus and summarize the cross-species usefulness of 20 microsatellite loci. The novel microsatellite loci for Cx. quinquefasciatus were isolated using an enrichment protocol (Keyghobadi et al . 2004). Fourteen percent of the colonies enriched for (GT) X were found to have GT repeats, whereas only 4% of those enriched for (GA) X had GA repeats. Primers were designed to amplify 13 of the 29 microsatellite-containing fragments; the remaining fragments had insufficient flank or extremely long microsatellites (> 25 repeats) that are difficult to score. We successfully optimized primers for five (GT) X -containing and one (GA) X -containing microsatellite loci (Table 1). In addition, from a separate protocol for microsatellite development targeting Plasmodium relictum using infected Correspondence: D. M. Fonseca, Present address: Patrick Center for Environmental Research, Academy of Natural Sciences, 1900 Benjamin Franklin Pkwy, Philadelphia, PA, 19103, USA. Fax: +215 299 1182; E-mail: fonseca@acnatsci.org §Present address: Department of Biology, Okanagan University College, Kelowna, British Columbia, Canada. ¶Present address: Department of Biological Sciences, Lehigh University, 111 Research Drive Bethlehem, PA 18015, USA. ††Julie L. Smith and Nusha Keyghobadi contributed equally. Order decided by coin toss.