Sorawat Thongsahuan

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0% oocyst rates were obtained, in comparison to the 86.67-100% oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0% sporozoite rates were obtained, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09%, 6.67% and 11.76% sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67% and 64.29% sporozoite rates were obtained, respectively, in comparison to 90% sporozoite rates recovered from An. cracens.

Cytogenetic and molecular evidence for an additional new species within the taxon Anopheles barbirostris (Diptera: Culicidae) in Thailand

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 (X1, X2, Y1). 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 F1 progenies. Moreover, available F1 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 F1 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.

Susceptibility of eight species members in the Anopheles hyrcanus group to nocturnally subperiodic Brugia malayi

BackgroundFilariasis, caused by Brugia malayi, is a public health problem in Thailand. Currently, at least two locations in southern Thailand are reported to be active endemic areas. Two and four Mansonia species are primary and secondary vectors, respectively, of the nocturnally subperiodic race, whereas, Coquillettidia crassipes is a vector of the diurnally subperiodic race. Although several Anopheles species have been incriminated extensively as natural and/or suspected vectors of B. malayi, little is known about vector competence between indigenous Anopheles and this filaria in Thailand. Thus, the susceptibility levels of eight species members in the Thai An. hyrcanus group to nocturnally subperiodic B. malayi are presented herein, and the two main refractory factors that affect them in different degrees of susceptibility have been elucidated.MethodsAedes togoi (a control vector), An. argyropus, An. crawfordi, An. nigerrimus, An. nitidus, An. paraliae, An. peditaeniatus, An. pursati and An. sinensis were allowed to feed artificially on blood containing B. malayi microfilariae, and dissected 14 days after feeding. To determine factors that take effect at different susceptibility levels, stain-smeared blood meals were taken from the midguts of Ae. togoi, An. peditaeniatus, An. crawfordi, An. paraliae, An. sinensis and An. nitidus immediately after feeding, and their dissected-thoraxes 4 days post blood-feedings were examined consecutively for microfilariae and L1 larvae.ResultsThe susceptibility rates of Ae. togoi, An. peditaeniatus, An. crawfordi, An. nigerrimus, An. argyropus, An. pursati, An. sinensis, An. paraliae and An. nitidus to B. malayi were 70–95%, 70–100%, 80–85%, 50–65%, 60%, 60%, 10%, 5%, and 0%, respectively. These susceptibility rates related clearly to the degrees of normal larval development in thoracic muscles, i.e., Ae. togoi, An. peditaeniatus, An. crawfordi, An. paraliae, An. sinensis and An. nitidus yielded normal L1 larvae of 93.15%, 96.34%, 97.33%, 23.60%, 15.38% and 0%, respectively.ConclusionsAn. peditaeniatus, An. crawfordi, An. nigerrimus, An. argyropus and An. pursati were high potential vectors. An. paraliae and An. sinensis were low potential vectors, while An. nitidus was a refractory vector. Two refractory mechanisms; direct toxicity and/or melanotic encapsulation against filarial larval were involved in the refractoriness of development in the thoracic muscles of the mosquito.

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.

Genetic compatibility between Anopheles lesteri from Korea and Anopheles paraliae from Thailand

To assess differentiation and relationships between Anopheles lesteri and Anopheles paraliae we established three and five iso-female lines of An. lesteri from Korea and An. paraliae from Thailand, respectively. These isolines were used to investigate the genetic relationships between the two taxa by crossing experiments and by comparing DNA sequences of ribosomal DNA second internal transcribed spacer (ITS2) and mitochondrial DNA cytochrome c oxidase subunit I (COI) and subunit II (COII). Results of reciprocal and F1-hybrid crosses between An. lesteri and An. paraliae indicated that they were compatible genetically producing viable progenies and complete synaptic salivary gland polytene chromosomes without inversion loops in all chromosome arms. The pairwise genetic distances of ITS2, COI and COII between these morphological species were 0.040, 0.007-0.017 and 0.008-0.011, respectively. The specific species status of An. paraliae in Thailand and/or other parts of the continent are discussed.

Seasonal biodiversity of black flies (Diptera: Simuliidae) and evaluation of ecological factors influencing species distribution at Doi Pha Hom Pok National Park, Thailand

This is the first study on the seasonal biodiversity of black flies and evaluation of ecological factors influencing their distribution at Doi Pha Hom Pok National Park, northern Thailand. Larvae were collected from six fixed-stream sites in relation to altitude gradients from May 2011 to April 2013. The water temperature, water pH, conductivity, total dissolved solids (TDS), salt, water velocity, stream width and depth, streambed particle sizes, riparian vegetation, and canopy cover were recorded from each site. Monthly collections from the six sites yielded 5475 last-instar larvae, belonging to 29 black fly species. The most frequently found species from all sites were Simulium asakoae (100%) followed by Simulium yuphae (83.3%), and Simulium chiangdaoense, Simulium gombakense, Simulium phahompokense, Simulium fruticosum, Simulium maeaiense and Simulium fenestratum (66.6%). Of the 5475 last-instar larvae, S. maeaiense (19.3%), S. chiangdaoense (15.8%) and S. asakoae (14.8%), were the three most abundant species. The Shannon diversity index (H) at the six sites with different altitudes of 2100m, 2000m, 1500m, 1400m, 700m, and 500m above mean sea level, were 2.042, 1.832, 2.158, 2.123, 1.821 and 1.822, respectively. The Shannon index and number of taxa in the cold season were higher than those in the rainy and hot seasons. Principal component analysis (PCA) indicated that at least three principal components have eigen values >1.0 and accounted for 93.5% of the total variability of ecological factors among sampling sites. The Canonical correspondence analyses (CCA) showed that most species had a trend towards altitude, canopy cover, riparian vegetation and water velocity.

DNA barcoding for the identification of eight species members of the Thai Hyrcanus Group and investigation of their stenogamous behavior

Eight species members of the Thai Hyrcanus Group were identified based on the intact morphology and molecular analysis (COI barcoding, 658 bp) of F1-progenies. Five iso-female lines of each species were pooled in order to establish stock colonies. A stenogamous colony of each species was investigated by making 200 and 300 newly emerged adult females and males co-habit in a 30 cm cubic cage for one week. After ovipositon, the spermathecae of females were examined for sperms. The results revealed that Anopheles argyropus, Anopheles crawfordi, Anopheles nitidus, Anopheles pursati, Anopheles sinensis, Anopheles nigerrimus, Anopheles paraliae and Anopheles peditaeniatus yielded insemination rates of 0%, 0%, 0%, 31%, 33%, 42%, 50% and 77%, respectively. Continuous selection to establish stenogamous colonies indicated that An. sinensis, An. pursati, An. nigerrimus, An. paraliae and An. peditaeniatus provided insemination rates of 33-34%, 27-31%, 42-58%, 43-57% and 61-86% in 1, 2, 5, 6 and 20 generations of passages, respectively.

Comparative Studies on the Stenogamous and Eurygamous Behavior of Eight Anopheles Species of the Hyrcanus Group (Diptera: Culicidae) in Thailand.

Establishment of laboratory colony is essential for mosquito-borne-disease research. Mating behavior of stenogamous Anopheles peditaeniatus and seven eurygamous species (Anopheles argyropus, Anopheles crawfordi, Anopheles nigerrimus, Anopheles nitidus, Anopheles paraliae (=An. lesteri), Anopheles pursati and Anopheles sinensis), were investigated and compared in this study. The self-mating success of adult mosquitoes in different size cages at two density resting surface (DRS) values, 3.6 and 7.2, was statistically significant between stenogamous and eurygamous species. The results obtained from comparative measurements of specific characters in adult females (maxillary palpomere and antennal sensilla characters) and males (wing and genitalia) indicate those characters might influence the mating success of An. peditaeniatus in a small cage. The gonostylus of An. peditaeniatus was shorter than the eurygamous species. Additionally, the lower frequency of clasper movement and shorter mating time could be important mechanisms that control the stenogamous behavior of An. peditaeniatus. Interestingly, for the first time, a cluster of large sensilla coeloconica was recorded on the antenna of An. argyropus and An. peditaeniatus females. There was no statistically significant difference in the mean number per female of those large antennal sensilla coeloconica among six of the eurygamous species.

Development of a multiplex PCR assay for the identification of eight species members of the Thai Hyrcanus Group (Diptera: Culicidae)

The Hyrcanus Group comprises important vectors of malaria because of Plasmodium vivax Grassi and Feletti and filariasis caused by Brugia malayi Brug in many countries of South, Southeast and East Asian regions. In Thailand, eight species members (Anopheles argyropus Swellengrebel, An. crawfordi Reid, An. nigerrimus Giles, An. nitidus Harrison, Scanlon & Reid, An. paraliae Sandosham, An. peditaeniatus Leicester, An. pursati Laveran and An. sinensis Wiedemann) of the Hyrcanus Group have been recognized. Due to morphological overlap, adult females of the Hyrcanus Group in Thailand have been misidentified markedly among the eight species, particularly when using the traumatic scales of wild-caught specimens from epidemiology and control approaches. Therefore, this study first developed a simple and robust multiplex PCR assay, based on second internal transcribed spacer sequences of ribosomal DNA, for differentiating the eight species members of the Thai Hyrcanus Group.

Scanning electron microscopy of Anopheles hyrcanus group (Diptera: Culicidae) eggs in Thailand and an ultrastructural key for species identification.

The eggs of Anopheles argyropus, Anopheles crawfordi, Anopheles nigerrimus, Anopheles nitidus, Anopheles paraliae, Anopheles peditaeniatus, Anopheles pursati, and Anopheles sinensis are described with the aid of scanning electron micrographs. Comparisons of the egg structure among the eight species showed that the eggs differed with respect to the following characteristics: the deck—complete (An. argyropus, An. nigerrimus, An. paraliae, An. peditaeniatus, and An. sinensis); variable (complete, split and incomplete decks found together within an egg batch/An. crawfordi); and division into an area at each end (An. nitidus and An. pursati). The ratios of the entire length per maximal deck width within the area covered by floats were 3.33–6.86 (An. sinensis), 8.78–18.20 (An. peditaeniatus), 13.67–22 (An. nigerrimus), 26.33–44.25 (An. paraliae), and 26.99–75.94 (An. argyropus). The numbers of float ribs were 21–27 (An. peditaeniatus) and 28–34 (An. nigerrimus), and the total numbers of anterior and posterior tubercles were 6–8 (An. paraliae) and 9–11 (An. argyropus). Exochorionic sculpturing was of reticulum type (An. argyropus, An. crawfordi, An. nigerrimus, An. nitidus, An. paraliae, An. peditaeniatus, and An. sinensis) and pure tubercle type (An. pursati). Attempts are proposed to construct a robust key for species identification based on the morphometrics and ultrastructures of eggs under scanning electron microscopy.