Intraspecific hybridization of Anopheles sinensis (Diptera: Culicidae) strains from Thailand and Korea

Anopheles (Anopheles) sinensis [Wiedemann (1828)] is a member of the hyrcanus species group, and it has been incriminated as the natural or experimental malaria vectors in the Republic of Korea, Japan, China, and Indonesia. In Thailand, however, An. sinensis seems to be of little medical importance. Hybridization tests among the three iso-female lines (isolines) of An. sinensis [i.e., Form A (X, Y1) and Form B (X, Y2) (Thailand strain), and Form B (X, Y2) (Korean strain)] were established based on two distinct types of metaphase chromosomes and geographical differences The chromosomal form of the Korean strain was first identified from this study. Results of reciprocal and back crosses indicated that both karyotypic forms of theAn. sinensis Thailand and Korean strains were genetically compatible, and provided viable progenies and completely synaptic polytene chromosomes. The sequences of the rDNA internal-transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit II (COII) among the An. sinensis strains were nearly identical to each other, and the intraspecific sequence variability was very low (0.0-0.6%). Sequence comparisons among the cryptic inter-species (i.e., An. sinensis, An. lesteri, and An. yatsushiroensis), however, revealed extensive divergence, and the intraspecific variability ranged from 12.2 to 34.6%. Therefore, it is concluded from these results and previous vector ability studies that the An. sinensis Forms A and B exhibit cytological polymorphic races that have different vector abilities in their transmission of malaria, depending on their geographical locations.     

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.     

Prevalence of Haplorchis taichui and Haplorchoides sp. metacercariae in freshwater fish from water reservoirs, Chiang Mai, Thailand

A parasitological investigation on trematode metacercariae was made on 62 freshwater fishes of 13 species in northern Thailand; Cyclocheilichthys apogon, Puntioplites proctozysron, Labiobarbus siamensis, Barbodes gonionotus, Barbodes altus, Henicorhynchus siamensis, Osteochilus hasselti, Notopterus notopterus, Mystacoleucus marginatus, Anabas testudineus, Systomus orphoides, Morulius chrysophykadian, and Hampala macrolepidota. The fish were caught over the summer period (February-May 2007) from 2 Chiang Mai water reservoirs, i.e., the Mae Ngad (UTM 47Q E 503200, 47Q N 2119300) and the Mae Kuang Udomtara (UTM 47Q E 513000, 47Q N 2092600) Reservoirs in Chiang Mai province, Thailand. The prevalence of heterophyid (Haplorchis taichui and Haplorchoides sp.) metacercariae in these fish was 83.9% and 74.2% in the Mae Ngad and Mae Kuang Udomtara Reservoirs, respectively. The highest intensity of heterophyid metacercariae in H. siamensis in the Mae Ngad was 120.4 and that in P. proctozysron in the Mae Kuang Udomtara was 180.0. The fish, A. testudineus, C. apogon, and M. chrysophykadian, were not found to be infected with H. taichui metacercariae. The results show that the freshwater fish in Chiang Mai water reservoirs are heavily infected with H. taichui and Haplorchoides sp. metacercariae.     

Diversity of Anopheles species and trophic behavior of putative malaria vectors in two malaria endemic areas of northwestern Thailand

We determined the species diversity, blood‐feeding behavior, and host preference of Anopheles mosquitoes in two malaria endemic areas of Tak (Mae Sot District) and Mae Hong Son (Sop Moei District) Provinces, located along the Thai border with Myanmar, during a consecutive two‐year period. Anopheline mosquitoes were collected using indoor and outdoor human‐landing captures and outdoor cow‐baited collections. Mosquitoes were initially identified using morphological characters, followed by the appropriate multiplex AS‐PCR assay for the identification of sibling species within Anopheles (Cellia) complexes and groups present. Real‐time PCR was performed for parasite‐specific detection in mosquitoes (Plasmodium spp. and Wuchereria bancrofti). A total of 7,129 Anopheles females were captured, 3,939 from Mae Sot and 3,190 from Sop Moei, with 58.6% and 37% of all anophelines identified as An. minimus, respectively. All three malaria vector complexes were detected in both areas. One species within the Minimus Complex (An. minimus) was present along with two related species in the Funestus Group, (An. aconitus, An. varuna), two species within the Dirus Complex (An. dirus, An. baimaii), and four species within the Maculatus Group (An. maculatus, An. sawadwongporni, An. pseudowillmori, and An. dravidicus). The trophic behavior of An. minimus, An. dirus, An. baimaii, An. maculatus, and An. sawadwongporni are described herein. The highest An. minimus densities were detected from February through April of both years. One specimen of An. minimus from Mae Sot was found positive for Plasmodium vivax.     

Evidence for a conspecific relationship between two morphologically and cytologically different forms of Korean Anopheles pullus mosquito

Despite the medical importance of anopheline mosquitoes as vectors of Korean vivax malaria, differentiation between Korean anopheline mosquitoes by traditional morphological taxonomic criteria is difficult. An. yatsushiroensis is the second most common Anopheles mosquito species in Korea and a possible vector of Korean vivax malaria together with An. sinensis, the predominant anopheline species. Recently, An. yatsushiroensis has been declared a synonym of An. pullus, based on comparisons of egg morphology and adult progeny, although they differ in ecology and morphology. To verify the species status of these two ambiguous forms, we established isofemale lines of Korean An. pullus and An. yatsushiroensis (An. pullus form yatsushiroensis) mosquitoes and investigated their genetic relationship by metaphase karyotype analysis, comparing the DNA sequences of rDNA internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunits I (COI) and II (COII), and by hybridization experiments. Two isofemale lines had differently shaped X and Y chromosomes. However reciprocal crosses between them yielded viable progeny with completely synaptic salivary gland polytene chromosomes. DNA analyses also strongly supported their conspecificity. The two strains also showed great sequence similarity in the ITS2, COI and COII regions (variation rate = 0.0 to 0.8%). Based on these findings, we conclude that the two forms, though differing distinctly in morphological, cytological and ecological traits, remain interfertile.     

A seroepidemiological study of Japanese encephalitis and dengue virus infections in the Chiang Mai area, Thailand

As part of a virological and epidemiological survey of encephalitis in the Chiang Mai area, the neutralizing (N) antibody levels of healthy persons to Japanese encephalitis (JE) and dengue (DEN) type 1-4 viruses were examined. A total of 985 blood samples was collected by the filter paper method from subjects of nine age groups in five districts, four (Pasang, Sarapee, Doi Saket and Mae Taeng) in the Chiang Mai Valley and one (Fang) in another valley separated by several ranges of mountains from the Chiang Mai Valley. From analyses of the results of N tests on the specimens, the following conclusions were drawn about the prevalences of JE and DEN viruses in the Chiang Mai area: (1) In the Chiang Mai Valley, the percentage incidences of N antibodies to JE and DEN viruses increased with age and by the age of 15, two thirds or more of the residents had been infected with JE and all DEN viruses except DEN type 2 virus, which showed the lowest prevalence. (2) In the Fang district, the percentage incidence of N antibody to JE virus increased with age, but those to DEN viruses did not, indicating much lower prevalences in the past of all four serotypes of DEN viruses in this district than in the Chiang Mai Valley. (3) At present, most infants in the Chiang Mai area, including the Fang district, seem to be exposed to DEN viruses first and later to JE virus.     

Cytogenetic,hybridization and molecular evidence of four cytological forms of Anopheles nigerrimus (Hyrcanus Group) in Thailand and Cambodia

Thirteen isoline colonies of Anopheles nigerrimus were established from individual wild‐caught females collected from cow‐baited traps at locations in Thailand and Cambodia. Three types of X (X₁, X₂, X₃) and 4 types of Y (Y₁, Y₂, Y₃, Y₄) chromosomes were recovered, according to differing amounts of extra heterochromatin. Four karyotypic forms were designed depending upon apparently distinct figures of X and Y chromosomes, i.e., Form A (X₁, X₂, X₃, Y₁), B (X₂, X₃, Y₂), C (X₁, Y₃), and D (X₃, Y₄). Forms C and D were new metaphase karyotypes discovered in this study. Form A appeared to be common in both Thailand and Cambodia. Forms B and D were found to be rather specific to southern and northeastern Thailand, respectively, whereas Form C was confined to Cambodia. Hybridization experiments among the eight isoline colonies, which were representative of four karyotypic forms of An. nigerrimus, demonstrated genetic compatibility in giving viable progenies and synaptic salivary gland polytene chromosomes through F₂‐generations. These results elucidated the conspecific relationship, comprising four cytological forms within this taxon. Supportive evidence was confirmed further by very low intraspecific sequence variations (average genetic distance = 0.002–0.007) of the nucleotide sequences in ribosomal DNA [second internal transcribed spacer (ITS2)] and mitochondrial DNA [cytochrome c oxidase subunit I (COI) and subunit II (COII)].     

Molecular and morphological studies on the Anopheles minimus group of mosquitoes in southern China: taxonomic review,distribution and malaria vector status

Abstract. Mosquitoes of the Anopheles minimus group (Diptera: Culicidae) from nine Provinces of southern China were identified morphologically and by molecular characterization, using single‐strand conformation polymorphisms (SSCPs) and sequence data for the D3 region of the 28S ribosomal DNA and the mitochondrial COII locus. Species A and C (sensu Green et al., 1990 ) of the An. minimus complex were found to be sympatric in Yunnan Province. Species A occurs eastward from Yunnan through southern Guangxi, Hainan, Guangdong and Taiwan Provinces, whereas species C occurs northward to northern Guangxi, Guizhou and Sichuan Provinces. Morphological and molecular evidence (based on specimens from the field and four isofemale lines) shows that An. minimus forms A and B (sensu Yu & Li, 1984 ) are morphological variants of species A, which is accepted as An. minimus Theobald sensu stricto (type‐locality: Pokfulam, Hong Kong). The so‐called subspecies x of An. minimus (sensu Baba, 1950 ) is reinterpreted as An. aconitus Dönitz. The distribution and vector status of members of the An. minimus group are discussed in relation to the historical and current transmission of malaria and filariasis in China. Both An. minimus A and C have been implicated as widespread vectors of malaria, whereas only species A has been found in Hainan, where An. minimus s.l. was a vector of Bancroftian filariasis. The presence of An. aconitus in Hainan and Yunnan Provinces is confirmed, but the occurrence of An. varuna Iyengar and An. fluviatilis James, which were previously recorded in China, could not be verified.     

Molecular systematics of the genus Megoura (Hemiptera: Aphididae) using mitochondrial and nuclear DNA sequences

To construct the molecular systematics of the genus Megoura (Hemiptera: Aphididae), DNA based-identifi-cation was performed using four mitochondrial and three nuclear DNA regions: partial cytochrome c oxidase I (COI), partial tRNA-leucine + cytochrome c oxidase II (tRNA/COII), cytochrome b (CytB), partial 12S rRNA + tRNA-valine + 16S rRNA (12S/16S), elongation factor-1 alpha (EF1 alpha), and the internal transcribed spacers 1 and 2 (ITS1, ITS2). Pairwise sequence divergences between taxa were compared, and phylogenetic analyses were performed based on each DNA region separately, and the combined datasets. COI, CytB, EF1 alpha, ITS1, and ITS2 were relatively effective in determining species and resolving their relationships. By contrast, the sequences of tRNA/COII and 12S/16S were not able to separate the closely related species. CytB and EF1alpha gave better resolution with higher average sequence divergences (4.7% for CytB, 5.2% for EF1 alpha). The sequence divergence of COI (3.0%) was moderate, and those of the two ITS regions (1.8% for ITS1, 2.0% for ITS2) were very low. Phylogenetic trees were constructed by minimum evolution, maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses. The results indicated that the phylogenetic relationships between Megoura species were associated with their host preferences. Megoura brevipilosa and M. lespedezae living on Lespedeza were closely related, and M. nigra, monophagous on Vicia venosa, was rather different from M. crassicauda, M. litoralis, and M. viciae, which are oligophagous on Lathyrus and Vicia. The three populations of M. crassicauda formed a clade separated from M. litoralis and M. viciae. Nevertheless M. litoralis and M. viciae, which are morphologically similar, were not separated due to negligible sequence divergence. We discuss the phylogenetic relationships of the Megoura, and the usefulness of the seven DNA regions for determining the species level phylogeny of aphids.     

Cytogenetic,cross-mating and molecular evidence of four cytological races of Anopheles crawfordi (Diptera: Culicidae) in Thailand and Cambodia

Twenty-nine isolines of Anopheles crawfordi were established from wild-caught females collected from cow-baited traps in Thailand and Cambodia. Three types of X (X₁, X₂, X₃) and four types of Y (Y₁, Y₂, Y₃, and Y₄) chromosomes were identified, according to differing amounts of extra heterochromatin. These sex chromosomes represent four metaphase karyotypes, i.e., Forms A (X₁, X₂, X₃, Y₁), B (X₁, X₂, X₃, Y₂), C (X₂, Y₃) and D (X₂, Y₄). Forms C and D are novel metaphase karyotypes confined to Thailand, whereas forms A and B appear to be common in both Thailand and Cambodia. Cross-mating experiments between the four karyotypic forms indicated genetic compatibility in yielding viable progenies and synaptic salivary gland polytene chromosomes. The results suggest that the forms are conspecific and A. crawfordi comprises four cytological races, which is further supported by very low intraspecific variation (mean genetic distance = 0.000–0.018) of the nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA sequences (COI, COII).     

Mitochondrial DNA analyses reveal low genetic diversity in Culex quinquefasciatus from residential areas in Malaysia

The present study explored the intraspecific genetic diversity, dispersal patterns and phylogeographic relationships of Culex quinquefasciatus Say (Diptera: Culicidae) in Malaysia using reference data available in GenBank in order to reveal this species' phylogenetic relationships. A statistical parsimony network of 70 taxa aligned as 624 characters of the cytochrome c oxidase subunit I (COI) gene and 685 characters of the cytochrome c oxidase subunit II (COII) gene revealed three haplotypes (A1–A3) and four haplotypes (B1–B4), respectively. The concatenated sequences of both COI and COII genes with a total of 1309 characters revealed seven haplotypes (AB1–AB7). Analysis using tcs indicated that haplotype AB1 was the common ancestor and the most widespread haplotype in Malaysia. The genetic distance based on concatenated sequences of both COI and COII genes ranged from 0.00076 to 0.00229. Sequence alignment of Cx. quinquefasciatus from Malaysia and other countries revealed four haplotypes (AA1–AA4) by the COI gene and nine haplotypes (BB1–BB9) by the COII gene. Phylogenetic analyses demonstrated that Malaysian Cx. quinquefasciatus share the same genetic lineage as East African and Asian Cx. quinquefasciatus. This study has inferred the genetic lineages, dispersal patterns and hypothetical ancestral genotypes of Cx. quinquefasciatus.     

Development of molecular diagnostic markers for sharpshooters Homalodisca coagulata and Homalodisca liturata for use in predator gut content examinations

To aid in identifying key predators of Proconiini sharpshooter species present in California, we developed and tested molecular diagnostic markers for the glassy‐winged sharpshooter, Homalodisca coagulata (Say), and smoke‐tree sharpshooter, Homalodisca liturata (Ball) (Homoptera: Cicadellidae). Two different types of markers were compared, those targeting single‐copy sequence characterized amplified regions (SCAR) and mitochondrial markers targeting the multicopy cytochrome oxidase subunit genes I (COI) and II (COII). A total of six markers were developed, two SCAR and four mitochondrial COI or COII markers. Specificity assays demonstrated that SCAR marker HcF5/HcR7 was H. coagulata specific and HcF6/HcR9 was H. coagulata/H. liturata specific. COI (HcCOI‐F/R) and COII (HcCOII‐F4/R4) markers were H. coagulata specific, COII (G/S‐COII‐F/R) marker was H. coagulata/H. liturata specific, and lastly, COII marker (Hl‐COII‐F/R) was H. liturata specific. Sensitivity assays using genomic DNA showed the COI marker to be the most sensitive marker with a detection limit of 6 pg of DNA. This marker was 66‐fold more sensitive than marker Hl‐COII‐F/R that showed a detection limit of 400 pg of DNA. In addition, the COI marker was 4.2‐fold more sensitive than the COII marker. In predator gut assays, the COI and COII markers demonstrated significantly higher detection efficiency than the SCAR markers. Furthermore, the COI marker demonstrated slightly higher detection efficiency over the COII marker. Lastly, we describe the inclusion of an internal control (28S amplification) for predation studies performing predator gut analyses utilizing the polymerase chain reaction (PCR). This control was critical in order to monitor reactions for PCR failures, PCR inhibitors, and for the presence of DNA.     

Two new Kirschsteiniothelia species with Dendryphiopsis anamorphs cluster in Kirschsteiniotheliaceae fam. nov

Two new Kirschsteiniothelia species are proposed in this study; both were collected on decaying wood from Chiang Mai and Chiang Rai provinces in northern Thailand. The taxa were isolated and the morphological characters are described and illustrated. ITS, LSU and SSU combined sequence analysis showed taxa of Kirschsteiniothelia separating into three lineages: (i) K. elaterascus grouped within Morosphaeriaceae (Pleosporales); (ii) K. maritima clustered with Mytilinidion spp. as a sister group in the Mytilinidiaceae clade; and (iii) the two new Kirschsteiniothelia species, which produce Dendryphiopsis anamorphs in culture, clustered with K. aethiops (the generic type) and the anamorph D. atra. The new family Kirschsteiniotheliaceae is introduced to accommodate taxa grouping with K. aethiops. K. elaterascus is transferred to Morosphaeria (Morosphaeriaceae) and a new genus Halokirschteiniothelia is introduced to accommodate K. maritima (Mytilinidiaceae).     

A new species of Docosia Winnertz from Central Europe,with DNA barcoding based on four gene markers (Diptera,Mycetophilidae)

A new species of Docosia Winnertz, Docosia dentata sp. n., is described and illustrated, based on a single male specimen collected in Muránska planina National Park in Central Slovakia. DNA sequences (COI, COII, CytB, and ITS2) are included and compared for 13 species of Docosia. There was found only little congruence between the molecular results and previous scarce data about interspecific relationships based on morphology. The COI and CytB gene markers showed the highest interspecific gene distances while ITS2 showed the lowest ones. An updated key to the 23 Central European species of Docosia is also presented.     

Particulate matter,PM 10 & PM 2.5 levels,and airborne mutagenicity in Chiang Mai,Thailand

Daily levels of particulate matter (PM) in the ambient air (PM 2.5 and PM 10) were measured in a northern city of Thailand (Chiang Mai) from March 1998 to October 1999. Twenty-four-hour air particulate matter samples were collected each day with Airmetric Minivol portable air samplers. Monthly averages of PM 2.5 from four stations in Chiang Mai varied from 15.39 to 138.31microg/m(3) and 27.29 to 173.40 microg/m(3) for PM 10. The PM 2.5 annual average was 58.48 mg/m(3) and PM 10, 86.38 microg/m(3). Daily PM 2.5 (24h values) during the winter months in Chiang Mai frequently exceeded 200-300 microg/m(3). The maximum concentrations of PM 2.5 (24h average) in Chiang Mai air from December 1998 to April 1999 were 2.8-, 3.5-, 4.2-, 6.5- and 3.2-fold higher than the US Environmental Protection Agency (US EPA), PM 2.5, 24h standard of 65 microg/m(3). From May to October, the mean 24h levels of PM 2.5 and PM 10 were at acceptable levels. The data shows that during the winter season (December to March), levels of PM 2.5 and PM 10 in the Chiang Mai atmosphere are very high, and there may be significant health implications associated with these high concentrations. During the summer season, the fine particles were generally within the acceptable levels. To our knowledge, these are the first measurements of PM 2.5 to be reported for the city of Chiang Mai and they indicate considerable ambient fine particle exposures to the Chiang Mai population. In addition, dichloromethane extracts of airborne particulate matter PM 2.5 or PM 10 collected in the months of winter in the city of Chiang Mai were mutagenic to Salmonella typhimurium strain TA100 without metabolic activation. The mutagenicity appeared to track particle concentrations and increased in the presence of S9 mix.     

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.