Development of a Multiplexed Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Assay to Identify Common Members of the Subgenera Culex ( Culex ) and Culex ( Phenacomyia ) in Guatemala

and WNV has been isolated from Cx. ( Cux. ) quinquefasciatus Say and Cx. ( Cux. ) chidesteri Dyar in Guatemala (MoralesBetoulle and others, unpublished data) and detected in Cx. ( Cux. ) nigripalpus and Cx. ( Cux. ) interrogator Dyar and Knab in Chiapas, Mexico. 7 Additional WNV isolates in Guatemala were obtained from Cx. ( Cux. ) mosquito pools of uncertain species identification (Morales-Betoulle and others, unpublished data). Patois virus (Bunyaviridae) has also been isolated from Cx. ( Cux. ) thriambus Dyar in Mexico. 8 Identification of actual and potential arbovirus vectors in this subgenus is a major challenge to arbovirus ecology studies in this tropical ecosystem. Currently, 16 species in the subgenus Culex ( Culex ) and two species in the subgenus Culex ( Phenacomyia ) are known to occur in Guatemala. 9 Accurate morphological differentiation of these species is extremely difficult, requiring careful examination of larval skins from reared specimens and associated male genitalia. Identification of adult females is further complicated by overlapping morphological variation and by the lack of a current and comprehensive morphological key. Culex ( Cux. ) pseudostigmatosoma Strickman, Cx. ( Cux. ) restuans Theobald, and Cx. ( Phc. ) lactator Dyar and Knab were reported from Guatemala by Strickman and others 11 – 13 after the development of the morphological key by Clark-Gil and Darsie. 10 Additionally, confusion has arisen regarding the identification and nomenclature of some species. Strickman 14