Cloning and Sequence Analysis of the Circadian Clock GenesperiodandtimelessinAedes albopictus(Diptera: Culicidae)

Abstract

The genes period (per) and timeless (tim) are core components of the circadian clock that regulates a wide range of rhythmic biochemical, physiological, and behavioral processes in prokaryotes and eukaryotes. We used degenerate polymerase chain reaction (PCR) and Rapid Amplification of cDNA Ends (RACE) to clone and sequence the entire cDNAs of both the per and tim genes in Aedes albopictus (Skuse). We also used the 5' end of the Ae. albopictus per cDNA to identify previously unannotated sequence coding for the N-terminal region of the PERIOD protein in Aedes aegypti L. We sequenced genomic DNA of one mosquito from each of three geographically distinct populations (New Jersey, Florida, and Brazil), and identified three introns in the per gene and eight introns in the tim gene. Phylogenetic analyses and comparison of functional domains support the orthology of the newly identified per and tim genes. Analysis of nonsynonymous to synonymous substitution rates indicates that both the per and tim genes have evolved under strong selective constraint subsequent to the divergence ofAe. albopictus and Ae. aegypti. Taken together, these results provide resources that can be used to investigate the molecular genetics of circadian phenotypes in Ae. albopictus and other culicids, to perform comparative analyses of insect circadian clock function, and also to conduct phylogeographic analyses using single-copy nuclear introns.