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.

The symbiotic microorganisms of arthropod vectors are highly significant from several points of view, partly due to their possible roles in the transmission of pathogenic causative agents by blood-sucking vectors. Although ticks are well studied because of their significance to human health, novel microbial associations remain to be described. This review summarises several endosymbiotic bacterial species in hard ticks from various parts of the world, including Coxiella-, Francisella-, Rickettsia- and Arsenophonus-like symbionts as well as Candidatus Midichloria mitochondrii and Wolbachia. New methodologies for the isolation and characterization of tick-associated bacteria will, in turn, encourage new strategies of tick control by studying their endosymbionts.

Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods. They are associated with a variety of reproductive alterations in their hosts, the best known being cytoplasmic incompatability. The Wolbachia pipientis assemblage has been divided into two major groups (A and B) and 12 subgroups. We report herein the first systematic survey of Wolbachia in mosquitoes, and the first survey classifying Wolbachia infections by subgroup. Wolbachia were detected in 28.1% of 89 wild-caught mosquito species, based on a polymerase chain reaction assay using ftsZ and wsp gene primers. Infections were found in all major disease vector genera except Anopheles. Nine of the 12 Wolbachia subgroups were represented. Group B Wolbachia strains showed more phylogenetic concordance with their host taxa than group A strains. Of the 25 positive mosquito species, five were superinfected with group A bacteria strains (AA), eight were superinfected with A and B strains (AB), and one was superinfected with group B strains (BB). The widespread distribution of Wolbachia among mosquito species further supports their potential importance in the genetic control of disease vectors.

Intraspecific phylogeography has been used widely as a tool to infer population history. However, little attention has been paid to Southeast Asia despite its importance in terms of biodiversity. Here we used the cytochrome oxidase I gene of mitochondrial DNA (mtDNA) for a phylogeographic study of 147 individuals of the black fly Simulium tani from Thailand. The mtDNA revealed high genetic differentiation between the major geographical regions of north, east and central/south Thailand. Mismatch distributions indicate population expansions during the mid-Pleistocene and the late Pleistocene suggesting that current population structure and diversity may be due in part to the species' response to Pleistocene climatic fluctuations. The genealogical structure of the haplotypes, high northern diversity and maximum-likelihood inference of historical migration rates, suggest that the eastern and central/southern populations originated from northern populations in the mid-Pleistocene. Subsequently, the eastern region had had a largely independent history but the central/southern population may be largely the result of recent (c. 100,000 years ago) expansion, either from the north again, or from a relictual population in the central region. Cytological investigation revealed that populations from the south and east have two overlapping fixed chromosomal inversions. Since these populations also share ecological characteristics it suggests that inversions are involved in ecological adaptation. In conclusion both contemporary and historical ecological conditions are playing an important role in determining population genetic structure and diversity.

Separating the confounding effects of long-term population history from gene flow can be difficult. Here, we address the question of what inferences about gene flow can be made from mitochondrial sequence data in three closely related species of mosquitoes, Anopheles dirus species A, C, and D, from southeast Asia. A total of 84 sequences of 923 bp of the mitochondrial cytochrome oxidase I gene were obtained from 14 populations in Thailand, Myanmar, and Bangladesh. The genealogy of sequences obtained from two populations of AN: dirus C indicates no contemporary gene flow between them. The F(ST) value of 0.421 therefore probably represents a recent common history, perhaps involving colonization events. Anopheles dirus A and D are parapatric, yet no differentiation was seen either within or between species. The starlike genealogy of their haplotypes, smooth unimodal mismatch distributions, and excess of low frequency mutations indicate population expansion in An. dirus A and D. This, rather than widespread gene flow, explains their low within-species F(ST) values (0.018 and 0.022). The greater genetic diversity of An. dirus D suggests that expansion occurred first in species D and subsequently in species A. The current geographical separation and low hybrid fitness of these species also argue against ongoing interspecific gene flow. They suggest instead either historical introgression of mtDNA from An. dirus D into species A followed by independent range expansions, or a selective sweep of mtDNA that originated in An. dirus D. While not excluding contemporary gene flow, historical population processes are sufficient to explain the data in An. dirus A and D. The genealogical relationships between haplotypes could not be used to make inferences of gene flow because of extensive homoplasy due to hypervariable sites and possibly also recombination. However, it is concluded that this approach, rather than the use of fixation indices, is required in the future to understand contemporary gene flow in these mosquitoes. The implications of these results for understanding gene flow in another important and comparable group of malaria vector mosquitoes in Africa, the An. gambiae complex, are also discussed.