Species-specific primers in multiplex PCR for rapid identification of the biting midge <i>Forcipomyia taiwana</i> (Diptera: Ceratopogonidae)

Forcipomyia taiwana (FT) is a tiny hematophagous midge widely distributed in urban and suburban areas of Taiwan and southern China. Its high population densities cause intense itching, skin inflammation, and allergic reactions, posing concerns for public health, tourism, and local economies. Rapid and accurate identification among species, sexes, and life stages is essential for ecological studies and pest management. We developed 10 species-specific primer sets targeting the hypervariable internal transcribed spacer (ITS) region for rapid identification of F. taiwana. All primer sets successfully distinguished F. taiwana from other ceratopogonids across four genera and 18 congeneric species. The primers also consistently amplified DNA from 32 F. taiwana specimens collected from 12 localities in Taiwan, indicating minimal influence of intraspecific ITS variation. Each primer pair provided reliable diagnostic results and can be applied independently. The FT-specific primers offer a simple, rapid, and robust molecular tool that reduces processing time from two days to 3-4 h and eliminates the need for sequencing. These assays will facilitate ecological monitoring and support integrated pest management programs for F. taiwana.