Identification of five species of the Anopheles dirus complex from Thailand, using allele‐specific polymerase chain reaction

Abstract

The Anopheles dirus complex of mosquitoes contains some of the most important vectors of malaria in Southeast Asia. To distinguish five species of the complex that occur in Thailand, a method using the polymerase chain reaction (PCR) was developed. The method utilizes allele-specific amplification to detect fixed differences between the species in the DNA sequence of the ribosomal DNA internal transcribed spacer 2. Primers were designed to amplify fragments of diagnostic length from the DNA of the different species. The method was tested on 179 mosquitoes of the An. dirus complex from many parts of Thailand and shown to be effective. Every specimen was unambiguously identified as species A, B, C, D or F (i.e. An. dirus s.s. species B, C, D or An. nemophilous, respectively) by the PCR method, with confirmation of 58/61 identifications from polytene chromosome characteristics. For the other three specimens (3/44 from Kanchanaburi 5 locality), there was disagreement between the PCR and chromosomal methods of species identification (probably due to errors in the chromosomal identifications). Primers can be combined in a single PCR reaction providing a rapid, sensitive and straightforward method of species identification. Only small quantities of DNA are required, leaving most of the mosquito to be used for other analyses.

Comparative Human Landing Catch and CDC Light Trap in Mosquito Sampling in Knowlesi Malaria Endemic Areas in Peninsula Malaysia

“…An. cracens from all localities was subjected for molecular analysis based on inter-transcribed spacer region 2 (ITS2) of ribosomal DNA [11]- [13] in order to confirm the species. Figure 3 showed that those mosquitoes were confirmed as An.…”

Bacterial Biodiversity in Midguts of Anopheles Mosquitoes, Malaria Vectors in Southeast Asia

“…Anopheles mosquitoes were morphologically sorted by taxon before using specific AS-PCR assays for species identification within the complex or the group [20][21][22]. Each individual was split in two pieces, head-thorax for species identification and abdomen for midgut bacteria analysis.…”

“…In Southeast Asia, Anopheles vectors belong to species complexes with different involvement in the transmission of pathogens [19]. Few sibling species of the Dirus and Minimus Complexes and the Maculatus Group are involved in malaria and BLF, but specific role of each sibling species and factors influencing this role have never been studied due to the lack of reliable methods for species identification, now available [20][21][22]. As mosquito microbiota is one of the factors influencing pathogen transmission, this chapter is presenting the biodiversity of bacteria in the midgut of field-collected adults of 10 Anopheles species, topic less studied compared to the large number of studies presenting bacteria in the defense against parasites in laboratory conditions.…”

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

“…dirus B, which is one of the important malaria vectors, has been studied in many areas, for example, molecular studies of insect glutathione S-transferases 26,27,32,33,34 , genetic studies of Anopheles species complexes 2,13,49,50,51 , and malaria transmission 6,9,19,39,42 . However, little is known about the salivary gland proteins of this mosquito species.…”