PCR-based identification of Culex pipiens complex collected in Japan.
The Culex pipiens complex consists of vector mosquitoes that transmit important human pathogens. In this study we established a simplified method to distinguish three members of the Cx. pipiens complex, Cx. p. pallens Coquillet, Cx. p. form molestus Forskal, and Cx. quinquefasciatus Say, collected in Japan. Sequence analysis of the Drosophila Ace-orthologous acetylcholinesterase (Ace) gene (668 to 680 bp) revealed that a single polymorphic region characterizes each species. Based on this region, specific primers that distinguish Cx. p. form molestus (ACEpip2) and Cx. p. pallens (ACEpall2) were newly designed. Polymerase chain reactions were performed with the genomic DNA of Culex mosquitoes as the template, and these primers clearly distinguished two Culex spp. The accuracy of the designed primers was evaluated with 38 colonies of mosquito samples collected from 9 prefectures of Japan. The testing revealed that the distribution of anautogenous Cx. p. pipiens has not been confirmed in Japan. It also revealed that the male of Cx. p. pallens possesses an Ace gene haplotype that is highly similar to the sequence of Cx. quinquefasciatus. This improved method allows the evaluation of vector competence of Cx. p.molestus, which is the suspected vector of West Nile virus.
Kasai
Shinji
S
Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan. kasacin@nih.go.jp
Komagata
Osamu
O
Tomita
Takashi
T
Sawabe
Kyoko
K
Tsuda
Yoshio
Y
Kurahashi
Hiromu
H
Ishikawa
Takeshi
T
Motoki
Mitsugu
M
Takahashi
Tomoya
T
Tanikawa
Tsutomu
T
Yoshida
Masahiro
M
Shinjo
Goro
G
Hashimoto
Tomoyuki
T
Higa
Yukiko
Y
Kobayashi
Mutsuo
M
D000110:Acetylcholinesterase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D003465:Culex / Q000145:classification* / Q000235:genetics; D017931:DNA Primers; D019476:Insect Proteins / Q000235:genetics; D007564:Japan; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D017931:DNA Primers; D019476:Insect Proteins; D000110:Acetylcholinesterase