The Analysis of Genetic Polymorphism on Mitochondrial Hypervariable Region III in Thai Population.

Mitochondrial DNA (mtDNA) analysis is a genetic marker for human identification, especially matrilineal inheritance. Hypervariable regions (HVR) I and II of mtDNA have been currently performed for human identification worldwide. Further examination of HVRIII has been conducted with the aim of enhancing the power of discrimination. The aim of this research is to provide informative data on the polymorphisms of HVRIII in the Thai population in order to establish a national database for human identification. Thai people who were unrelated through the maternal lineage were recruited for blood collections. The mtDNA was extracted by Chelex extraction, amplified by polymerase chain reaction, and analyzed using Sequencing Analysis Software. The most common mutation in HVRIII was base substitution, followed by deletion and insertion. We discovered 40 unique haplotypes, with haplotype 489C being the most frequent. The haplotype diversity, power of discrimination, and random match probability were 0.8014, 0.7987, and 0.2013, respectively. Five-CA repeats were the most frequently observed in nucleotide positions 514-523. Our database can be employed as supplementary markers in addition to nuclear deoxyribonucleic acid (DNA) markers in forensic investigations. Moreover, the data could potentially enhance genetic identification and anthropological genetics research in Thailand.

Community Structure, Biodiversity and Spatiotemporal Distribution of the Black Flies (Diptera: Simuliidae) Using Malaise Traps on the Highest Mountain in Thailand.

Black flies form a group of small blood-sucking insects of medical and veterinary importance. This study aimed to investigate the community structure, biodiversity and spatial and temporal distribution of adult black flies in tropical rain forests, by using malaise traps in Doi Inthanon National Park, northern Thailand. Malaise traps were placed along six elevational gradients (400 m to 2500 m, above sea level) at Doi Inthanon National Park, Chiang Mai province, from December 2013 to November 2014. A total of 9406 adult female black flies belonging to five subgenera-Daviesellum (2%), Gomphostilbia (23%), Montisimulium (11%), Nevermannia (16%) and Simulium (48%)-were collected. Among 44 taxa found, S. tenebrosum complex had the highest relative abundance (11.1%), followed by the S. asakoae species-group (9.6%), the S. striatum species-group (7.7%), S. inthanonense (6.6%), S. doipuiense complex (6.4%), S. chomthongense complex (5.3%), S. chumpornense (5.1%) and S. nigrogilvum (4.1%). Two human-biting species-S. nigrogilvum and species in the S. asakoae species-group-were found in all of the collection sites with 100% species occurrence. Species richness was highest at mid elevation (1400 m), which is represented by 19 black fly species. The peak and lowest seasonal abundance was observed in the rainy and hot season, respectively. Seasonal species richness was highest in the cold season, except for that from elevation sites at 700 m, 1700 m and 2500 m. This study revealed that the malaise trap is effective in providing important data for further monitoring of the effects of environmental changes and conservation planning on the biodiversity of black flies in Doi Inthanon National Park.

The first molecular identification of benzimidazole resistance in Haemonchus contortus from goats in Thailand.

BACKGROUND AND AIM: Haemonchus contortus is one of the major trichostrongyloid nematodes affecting small ruminant production worldwide, especially in tropical and subtropical regions. Adult H. contortus suck the blood from the host abomasum leading to anemia and often death in heavily infected animals. The mainstay of parasitic control is an anthelmintic drug, but long-term drug use may cause drug resistance. The aim of this study was to examine benzimidazole resistance in H. contortus of goats from different regions in Thailand by detecting the frequency of the F200Y polymorphism in the ß-tubulin isotype 1 gene. MATERIALS AND METHODS: A total of 121 H. contortus adults were obtained from 31 naturally infected out of 37 slaughtered goats from city abattoirs in five regions of Thailand. The frequency of the F200Y polymorphism in the ß-tubulin isotype 1 gene was detected following the allele-specific polymerase chain reaction protocol. RESULTS: The overall genotype frequencies in Thailand were homozygous resistant (RR: 24%), heterozygous (SR: 44.6%), and homozygous susceptible (SS: 31.4%). The allele frequencies were resistant allele (R: 46%) and susceptible allele (S: 54%). The R allele frequency and the RR genotype varied from 30% to 65% and 0% to 43.9%, respectively. The frequency of R alleles was significantly higher in the southern region (0.65) as compared to northern (0.30, p=0.001), western (0.38, p=0.04), and central regions (0.30, p=0.03). The RR genotype was also significantly higher in the southern region (43.9%) versus the northern (0 %, p=0.001), western (11.8%, p=0.012), and central regions (17.4%, p=0.001). CONCLUSION: This is the first study of the detection of single-nucleotide polymorphisms in codon 200 of the ß-tubulin isotype 1 gene of H. contortus from goats in Thailand. These findings are essential and imply that an integrated approach is needed for issues such as drug treatment, farm management, prevention, and control strategies. This is of interest to farmers, veterinarians, and the department of livestock.

Hematological profile of blood parasitic infected dogs in Southern Thailand.

BACKGROUND AND AIM: Tick-borne pathogens such as Babesia canis, Hepatozoon canis, and Ehrlichia canis can cause serious disease in canines. Each blood parasite can be associated with different hematological characteristics in infected dogs. Identification of hematological alterations during routine laboratory screening of blood samples from dogs displaying clinical signs is essential for diagnosing blood parasitic infections. This study aimed to evaluate parasitic infections and hematological alterations in blood samples of infected dogs in Southern Thailand. MATERIALS AND METHODS: A total of 474 blood samples were collected from dogs presented at the Veterinary Teaching Hospital of the Prince of Songkla University between 2016 and 2019. An automatic hematology analyzer was used to establish hematological values; peripheral blood films were screened for blood parasites and their detection was associated with hematological alterations to determine the odds ratio (OR). RESULTS: This study found that E. canis (n=127) was the most common blood parasite infecting dogs in southern Thailand, followed by H. canis (n=100) and B. canis (n=24). Hematological alterations caused by Ehrlichia infections included anemia, thrombocytopenia, monocytosis, and eosinophilia (OR=14.64, 17.63, 20.34, and 13.43, respectively; p<0.01). The blood samples of Hepatozoon-infected dogs were characterized by anemia, thrombocytopenia, leukocytosis, neutrophilia, and monocytosis (OR=6.35, 3.16, 12.80, 11.11, and 17.37, respectively; p<0.01). Anemia, thrombocytopenia, eosinopenia, and lymphopenia (OR=10.09, 33.00, 20.02, and 66.47 respectively; p<0.01) were associated with B. canis-infected dogs. CONCLUSION: These data support the fact that hematological abnormalities are a hallmark for the identification of tick-borne infections. The hematological values, hereby reported, can be used as a guideline for the clinical diagnosis of canine blood parasitic infections in Southern Thailand.

Precision and accuracy of the Mindray BC-5000Vet hematology analyzer for canine and feline blood.

BACKGROUND: The Mindray BC-5000Vet hematology analyzer is a flow cytometry-based automated hematology analyzer that generates a complete blood count with a five-part white blood cell (WBC) differential count. OBJECTIVES: We aimed to validate reliability results of the Mindray BC-5000Vet for use in dog and cat blood. METHODS: Imprecision was performed using the manufacturer's quality control material at low, normal, and high levels. Blood sample results of healthy and ill dogs and cats were studied for comparability between manual methods and the Mindray BC-5000Vet analyzer. Forty dogs and 40 cats were included in the study. RESULTS: Precision for red blood cell (RBC) parameters was excellent, with a coefficient of variation within-run (%CVw ) and between-run (%CVb ) of <2%. WBC count and differential count showed %CVw and %CVb <8%; however, %CVw and %CVb of low-level control material gave >9% eosinophils. The correlation between the BC-5000 Vet and manual methods in normal and abnormal canine and feline blood samples showed excellent correlations for the RBC counts, hemoglobin concentrations, hematocrits, and WBC counts (r > .93). The differential WBC analysis of canine blood showed good correlation (r = .80-.92). Feline blood samples showed excellent correlations for neutrophils and lymphocytes, with a good correlation for monocytes and eosinophils. CONCLUSIONS: The Mindray BC-5000Vet hematology analyzer proved a suitable instrument for routine analysis in dogs and cats with various hematologic abnormalities.

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.

Biodiversity, seasonal abundance, and distribution of blackflies (Diptera: Simuliidae) in six different regions of Thailand.

BACKGROUND: Blackflies are an important medical and veterinary group of small blood-sucking insects. Ninety-three blackfly species have been reported in Thailand. However, information on their biodiversity and population dynamics in each region is lacking. The main aim of this study was to assess the regional biodiversity, seasonal abundance and distribution of blackflies in six eco-geographically different regions in the country. METHODS: Blackfly larvae and pupae were sampled monthly from 58 sites between May 2011 and April 2013. Diversity parameters, seasonal abundance, regional distribution and frequency of species occurrence in stream sites were analyzed. RESULTS: A total of 19,456 mature larvae representing 57 species, and belonging to six subgenera in the genus Simulium Latreille (s.l.), were found. The five predominant taxa were S. fenestratum (8.6%), the S. asakoae complex (8.3%), S. nakhonense (7.5%), the S. siamense complex (7.4%) and the S. doipuiense complex (6.7%). The most frequent taxa at all sites were the S. asakoae complex (84.5%), followed by S. fenestratum (82.8%), the S. siamense complex (75.9%), S. decuplum (60.3%), S. nakhonense (58.6%) and the S. tani complex (48.3%). The richness of regional species was highest (40 species) in the north and predominated in the cold season. However, blackflies in the south predominated during the hot season. The highest numbers of blackflies collected from central, northeastern, eastern and western regions of the country were observed in the rainy season. Overall, the mean number of blackflies collected across the six regions during the rainy and cold season had no statistically significant difference, but it differed significantly in the hot season. CONCLUSIONS: Blackflies in Thailand were surveyed in all three seasons across six geographical regions. These findings demonstrated that blackfly communities at each stream site varied with seasonality, and the regional relative abundance of blackflies differed markedly in the hot season. It was also found that the occurrence and distribution of blackflies in each region were associated strongly with elevation.

Natural Plasmodium vivax infections in Anopheles mosquitoes in a malaria endemic area of northeastern Thailand.

There was recently an outbreak of malaria in Ubon Ratchathani Province, northeastern Thailand. In the absence of information on malaria vector transmission dynamics, this study aimed to identify the anopheline vectors and their role in malaria transmission. Adult female Anopheles mosquitoes were collected monthly by human-landing catch in Na Chaluai District of Ubon Ratchathani Province during January 2014-December 2015. Field-captured mosquitoes were identified to species using morphology-based keys and molecular assays (allele-specific polymerase chain reaction, AS-PCR), and analysed for the presence of Plasmodium falciparum and Plasmodium vivax using an enzyme-linked immunosorbent assay (ELISA) for the detection of circumsporozoite proteins (CSP). A total of 1,229 Anopheles females belonging to 13 species were collected. Four anopheline taxa were most abundant: Members of the Anopheles barbirostris complex, comprising 38% of the specimens, species of the Anopheles hyrcanus group (18%), Anopheles nivipes (17%) and Anopheles philippinensis (12%). The other nine species comprised 15% of the collections. Plasmodium infections were detected in two of 668 pooled samples of heads/thoraces, Anopheles dirus (1/29) and An. philippinensis (1/97). The An. dirus pool had a mixed infection of P. vivax-210 and P. vivax-247, whereas the An. philippinensis pool was positive only for the latter protein variant. Both positive ELISA samples were confirmed by nested PCR. This study is the first to incriminate An. dirus and An. philippinensis as natural malaria vectors in the area where the outbreak occurred. This information can assist in designing and implementing a more effective malaria control programme in the province.

Scanning electron microscopy of antennal sensilla of the eight Anopheles species of the Hyrcanus Group (Diptera: Culicidae) in Thailand.

Antennal sensilla were first investigated in the eight medically and veterinary important Anopheles mosquito species (Anopheles argyropus, Anopheles crawfordi, Anopheles nigerrimus, Anopheles nitidus, Anopheles paraliae (= Anopheles lesteri), Anopheles peditaeniatus, Anopheles pursati, and Anopheles sinensis) of the Hyrcanus Group in Thailand, using scanning electron microscopy (SEM). Four types of sensilla, including sensilla chaetica (large and small), sensilla trichodea (sharp- and blunt-tipped), sensilla basiconica or grooved pegs (types I, II, and III), and sensilla coeloconica (large and small), were observed on the female antennae of the eight species. The greatest number of sensilla found along the flagellum of all the Anopheles species consisted of sensilla trichodea. Grooved pegs type II were not found on the antennae of An. peditaeniatus. Interestingly, clusters of 10-15 grooved pegs type III, with blunt-tipped and unevenly grooved-lengthwise sensilla, and a sunken group of 7-12 grooved pegs type III, with slightly curved and point-tipped sensilla, were found distally on flagellomeres 3-7 of An. argyropus and An. peditaeniatus, respectively. In addition, the key for species identification, based on fine structure and morphometrics of antennal sensilla among the eight species, was constructed and differentiated successfully. However, in order to focus intensively on the exact function of these sensilla, further electrophysiological study is needed in understanding their significant role in mosquito behavior, especially when these insects seek hosts for transmitting pathogens to humans.

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.

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.

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).

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.

Susceptibility of five species members of the Korean Hyrcanus Group to Brugia malayi, and hybridization between B. malayi-susceptible and -refractory Anopheles sinensis strains.

Five species members of the Korean Hyrcanus Group: Anopheles pullus, Anopheles sinensis, Anopheles kleini, Anopheles belenrae, and Anopheles lesteri were tested for susceptibility to Brugia malayi. They were allowed to feed artificially on blood containing B. malayi microfilariae and dissected 14 days after feeding. The susceptibility rates were 60%, 65%, 90%, 100% and 100% in An. pullus, An. sinensis, An. kleini, An. belenrae, and An. lesteri, respectively. As determined by levels of susceptibility, results indicated that An. pullus was a moderate potential vector, while An. sinensis, An. kleini, An. belenrae, and An. lesteri were high potential vectors, when compared with the 90-95% susceptibility rates of an efficient control vector, Ochlerotatus (=Aedes) togoi. An introgressive study of B. malayi-susceptible/-refractory genes was performed intensively by hybridization experiments between a high (Korean strain) and a low (Thailand strain) potential An. sinensis vectors. The susceptibility rates of F1-hybrids and backcross progenies were compared with parental stocks. The results indicated that the B. malayi-susceptible genes could be introgressed from a high to low potential An. sinensis vector by increasing the susceptibility rates from 0-5% in the parental stocks to 55% and 70% in F1-hybrids and backcross progenies, respectively. The increase of susceptibility rates related clearly to the increase of normal larval development in the thoracic muscles of F1-hybrids and backcross progenies.

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.

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.

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.

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.

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

BACKGROUND: Filariasis, 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. METHODS: Aedes 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. RESULTS: The 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. CONCLUSIONS: An. 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.

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)].