Seventeen isolines of Anopheles barbirostris derived from animal-biting female mosquitoes showed three karyotypic forms: Form A (X2, Y1) in five isolines from Phetchaburi province; Form B (X1, X3, Y2) in three and eight isolines from Chiang Mai and Ubon Ratchathani provinces, respectively; Form C (X2, Y3) in one isoline from Phetchaburi province. All 17 isolines exhibited an average branch summation of seta 2-VI pupal skins ranging from 12.1-13.0 branches, which was in the limit of A. barbirostris (6-18 branches). Of the 12 human-biting isolines from Chiang Mai province, five isolines showed Form B (X2, Y2), and seven isolines exhibited a new karyotypic form designated as Form E (X2, Y5). All of 12 isolines had an average branch summation of seta 2-VI pupal skins ranging from 22.4-24.5 branches, which was in the limit of Anopheles campestris (17-58 branches). Thus, they were tentatively designated as A. campestris-like Forms B and E. Hybridization between A. campestris-like Forms B and E showed that they were genetically compatible, yielding viable progeny for several generations suggesting conspecific relationships of these two karyotypic forms. Reproductive isolation among crosses between A. campestris-like Form B and A. barbirostris Forms A, B, and C strongly suggested the existence of these two species. In addition, the very low intraspecific variation (genetic distance<0.005) of the nucleotide sequence of ITS2 of the rDNA and COI and COII of mitochondrial DNA of the seven isolines of A. campestris-like Forms B and E supported their conspecific relationship. The large sequence divergence of ITS2 (0.203-0.268), COI (0.026-0.032), and COII (0.030-0.038) from genomic DNA of A. campestris-like Forms B and E and the A. barbirostris Forms A, B, and C clearly supported cytogenetic and morphological evidence.

Nine isoline colonies of Anopheles barbirostris Form A, derived from individual isofemale lines from Chiang Mai, Phetchaburi, and Kanchanaburi, were established in our insectary at Chiang Mai University. All isolines shared the same mitotic karyotype (X(1), X(2), Y(1)). Molecular analysis of deoxyribonucleic acid (DNA) sequences and polymerase chain reaction (PCR) products of ITS2, COI, and COII regions revealed three distinct groups: A1 (Chiang Mai), A2 (Phetchaburi), and A3 (Kanchanaburi). Crossing experiments among the three groups exhibited strong reproductive isolation, producing low and/or non-hatched eggs, and inviable and/or abnormal development of the reproductive system of F(1)-progenies. Asynaptic regions along the five polytene chromosome arms of F(1)-hybrid larvae clearly supported the existence of three sibling species within A. barbirostris Form A, provisionally named species A1, A2, and A3.

BackgroundMutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale.MethodsA multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR).ResultsThe results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory.ConclusionsOur multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.

ITS2 DNA sequences of 42 isoline colonies of Anopheles barbirostris species A1 and A2 were analyzed and a new genetic species, temporarily designated as species A4 (Chiang Mai), was revealed. The large sequence divergences of the ITS2 (0.116-0.615), COI (0.023-0.048), and COII (0.030-0.040) genes between A. barbirostris species A4/A1 (Chiang Mai), A4/A2 (Phetchaburi), A4/A3 (Kanchanaburi), and A4/Anopheles campestris-like Form E (Chiang Mai) provided good supporting evidence. Species A1, A2, A3, and A4 share a mitotic karyotype of Form A (X(1), X(2), Y(1)). Crossing experiments between species A4 and the other four species yielded strong reproductive isolation producing few and/or non-hatched eggs and inviable and/or abnormal development of the reproductive system of F(1) progenies. Moreover, available F(1) hybrid larvae showed asynaptic polytene chromosome arms. Hence, molecular and cytogenetic evidence strongly support the existence of A. barbirostris species A4, which is more closely related to A. campestris-like Form E than to species A1, A2, and A3. Additionally, crossing experiments among 12 and seven isolines of different cytological forms of species A1 (A, B, C, D) and A2 (A, B), respectively, yielded fertile and viable F(1) progenies. Thus, different karyotypic forms occurring in natural populations of species A1 and A2 merely represent intraspecies variation of sex chromosomes due to the extra blocks of heterochromatin.

Following the recent emergence of malaria in South Korea, vector control has been an important task. For this, vector identification is very important. Earlier, two PCR-based assays have been described. But, poor species resolution and their ability to include only 4-5 species limit their use. Thus, it has now become important to revise the assay identifying these members. In this study, a new assay based on internal transcribed spacer 2 and 28S of ribosomal DNA has been described. The assay successfully identified all the Korean malaria vector mosquitoes. Therefore, it is an indispensable tool to study ecology, abundance and biology of these species.