A multiplex PCR-based molecular identification of five morphologically related, medically important subgenus stegomyia mosquitoes from the genus Aedes (Diptera: Culicidae) found in the Ryukyu Archipelago, Japan

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Internal transcribed spacer regions of ribosomal DNA were sequenced, and new species-specific primers were designed to simplify the molecular identification of five morphologically related subgenus Stegomyia mosquito species-Aedes aegypti, Ae. albopictus, Ae. riversi, Ae. flavopictus, and Ae. daitensis-found in the Ryukyu Archipelago, Japan. Each newly designed primer was able to amplify a species-specific fragment with a different size. Conditions for multiplex PCR were optimized to identify all five species in a single PCR. This method is a convenient tool for entomological field surveys, particularly in arbovirus endemic/epidemic areas where some of these species coexist.