Systematic studies of Anopheles (Cellia) kochi (Diptera: Culicidae): Morphology, cytogenetics, cross-mating experiments, molecular evidence and susceptibility level to infection with nocturnally subperiodic Brugia malayi.

Anopheles kochi DÓ§nitz (Diptera: Culicidae) is a malaria vector in some countries in South and Southeast Asia. This is the first report to provide clear evidence that two different cytological forms of An. kochi are conspecific based on systematic studies. Two karyotypic forms, i.e., Form A (X1, X2, Y1) and a novel Form B (X1, X2, Y2) were obtained from a total of 15 iso-female lines collected from five provinces in Thailand. Form A was common in all provinces, whereas Form B was restricted to Ubon Ratchathani province. This study determined whether the two karyotypic variants of An. kochi exist as a single or cryptic species by performing cross-mating experiments in association with the sequencing of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA), and cytochrome c oxidase subunit I (COI) of mitochondrial DNA (mtDNA). Cross-mating experiments between the two karyotypic forms revealed genetic compatibility by providing viable progenies through F2 generations. The two forms showed a high sequence similarity of those two DNA regions (average genetic distances: ITS2 = 0.002-0.005, COI = 0.000-0.009). The phylogenetic trees based on ITS2 and COI sequences also supported that four strains (from Bhutan, Cambodia, Indonesia, and Thailand) were all of the same species. Five sensilla types housed on the antennae of female An. kochi were observed under scanning electron microscopy (SEM). In addition, this study found that An. kochi was a refractory vector, revealed by 0% susceptibility rates to infection with nocturnally subperiodic Brugia malayi. The cibarial armature was a resistant mechanism, as it killed the microfilariae in the foregut before they penetrated into the developmental site.

Evidence to support a conspecific nature of allopatric cytological races of Anopheles nitidus (Diptera: Culicidae) in Thailand.

Metaphase karyotype investigation on two allopatric strains of Anopheles nitidus Harrison, Scanlon, and Reid (Diptera: Culicidae) was conducted in Thailand during 2011-2012. Five karyotypic forms, i.e., Form A (X1, Y1), Form B (X1, Y2), Form C (X2, Y3), Form D (X1, X3, Y4), and Form E (X1, X2, X3, Y5) were obtained from a total of 21 isofemale lines. Forms A, B, and C were confined to Phang Nga Province, southern Thailand, whereas Forms D and E were restricted to Ubon Ratchathani Province, northeastern Thailand. Cross-mating experiments among the five isofemale lines, which were representative of five karyotypic forms of An. nitidus, revealed genetic compatibility by providing viable progenies and synaptic salivary gland polytene chromosomes through F2 generations. The results suggest that the forms are conspecific, and An. nitidus comprises five cytological races. The very low intraspecific sequence variations (average genetic distances = 0.002-0.008) of the nucleotide sequences in ribosomal DNA (internal transcribed spacer 2) and mitochondrial DNA (cytochrome c oxidase subunits I and II) among the five karyotypic forms were very good supportive evidence.

Karyotypic variation and geographic distribution of Anopheles campestris-like (Diptera: Culicidae) in Thailand

Seventy-one isolines of Anopheles campestris-like were established from wild-caught females collected from human-biting and animal-biting traps at 12 locations in Thailand. All isolines had an average branch summation of seta 2-VI pupal skins ranging from 20.3-30.0 branches, which is in the range of An. campestris (17-58 branches). They showed three different karyotypes based on the amount of extra heterochromatin in the sex chromosomes, namely Forms B (X2, Y2), E (X1, X2, X3, Y5) and a new karyotypic Form F (X2, X3, Y6). Form B has been found only in Chaing Mai and Kamphaeng Phet populations, while Forms E and F are widely distributed throughout the species range. Genetic crosses between the 12 isolines, which were arbitrarily selected as representatives of An. campestris-like Forms B, E and F, revealed genetic compatibility that provided viable progeny through F2 generations, suggesting a conspecific nature of these karyotypic forms. These results are supported by the very low intraspecies variation (genetic distance < 0.005) of ITS2, COI and COII from genomic DNA of the three karyotypic forms.