Abstract:

AIM: To distinguish cryptic species A and D of Anopheles dirus complex using polymerase chain reaction (PCR). METHODS: A diagnostic PCR assay of species was developed by use of three primers, one derived from highly conservative 5.8 S coding sequences and two from different interspecies sequence in the second internal transcribed spacer (ITS2) of ribosomal DNA. RESULTS: Using the PCR method, specific fragments were amplified in both species, the size of fragments is 374 bp for species A and 663 bp for species D. Thirty
samples of species A from AFRIMS and HN laboratory colony and seven samples of the species D from Yunnan Province were correctly identified by PCR. Satisfactory results were obtained from the amount of DNA as little as 1/1 600 of extracted DNA of a single mosquito or 1/5 of DNA derived from one leg of a mosquito triturated in water. A total of 148 field-collected specimens of Anopheles dirus from Heping (HP) , Baisha (BS) , Loukui(LK), and Maoyang (MY) in Hainan Province revealed fragment characteristic of species A, while 30 specimens from Mengla (ML) in Yunnan Province showed the specific fragment of species D. CONCLUSION: A simple and reliable Method was developed to identify cryptic species A and D of Anopheles dirus complex and it was further verified that Anopheles dirus from Hainan and Yunnan Provinces is the species A and the species D, respectively.

Key words: Polymerase chain reaction, Anopheles dirus, second internal transcribed spacer of ribosomal DNA