Differentiation of cryptic species A and D of Anopheles dirus complex by polymerase chain reaction

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.