Therapeutic trial of doxycyclin plus ivermectin for the treatment of Brugia malayi naturally infected cats.

Lymphatic filariasis (LF) is one of the neglected tropical diseases which causes permanent and long term disability worldwide. LF is caused by filarial nematode parasites, i.e. Wuchereria bancrofti, Brugia malayi, and B. timori. All available antifilarial drugs currently being used have shown a limited adulticidal activity. Discoveries of endosymbiont rickettsia-like bacterium, Wolbachia in filarial nematodes provided a novel approach for antibiotic use in eradication of filarial diseases. The earlier studies revealed the macrofilaricidal efficacy of doxycycline against filarial nematodes. Chemotherapeutic efficiency of doxycycline has been studied against many filarial parasites, but there are still no therapeutic trials of the drug regimens for B. malayi treatment in naturally infected cats. Thus, this study would be the first attempt to study the efficiency of doxycycline (DOXY) alone or in combination with ivermectin (IVM) for treatment of B. malayi in naturally infected cats. A total of 26 B. malayi-infected cats in the endemic areas were recruited and divided into 3 groups, receiving different treatment regimens; a single dose of ivermectin only (IVM), doxycycline only (DOXY) and a combination of ivermectin and doxycycline (DOXY-IVM). The efficacy of each therapatic regimen was evaluated by detecting the presence of microfilaria using parasitological and molecular techniques monthly up to 2 years after starting the treatment. The IVM treated group had a significant rapid reduction of microfilariae in the first month; however, recurrence of microfilaraemia was observed in some cats. By contrast, the DOXY and DOXY-IVM groups showed a better result with a gradual decrease in microfilariae with no recurrence. These 2 groups were not only virtually deprived of infection but also sustained the sterility of infection through the course of study. These results revealed the advantages of using in B. malayi treatment in cats. Doxycycline showed to have both microfilaricidal and adulticidal effects on lymphatic filariae which maintained the long-term response to control of B. malayi infection in cats.

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.

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.

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).

A new species and species-group of Simulium (Simulium) (Diptera: Simuliidae) from Thailand.

Simulium (Simulium) atipornae sp. nov. is described from females, males, pupae, and larvae in Thailand. This new species is characterized in the female by the claw with a small subbasal tooth, ovipositor valve triangular with its inner margin nearly straight; in the male by the style with a short subbasal protuberance and ventral plate Y-shaped, with toothed posterior margin; and in the pupa by the head and thoracic integument almost bare and gill with six filaments. Taxonomic notes are given to compare this new species with nine related species. A new species-group, the christophersi species-group, is proposed to accommodate S. atipornae sp. nov. and nine related species.

Identification and characterisation of Aedes aegypti aldehyde dehydrogenases involved in pyrethroid metabolism.

BACKGROUND: Pyrethroid insecticides, especially permethrin and deltamethrin, have been used extensively worldwide for mosquito control. However, insecticide resistance can spread through a population very rapidly under strong selection pressure from insecticide use. The upregulation of aldehyde dehydrogenase (ALDH) has been reported upon pyrethroid treatment. In Aedes aegypti, the increase in ALDH activity against the hydrolytic product of pyrethroid has been observed in DDT/permethrin-resistant strains. The objective of this study was to identify the role of individual ALDHs involved in pyrethroid metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Three ALDHs were identified; two of these, ALDH9948 and ALDH14080, were upregulated in terms of both mRNA and protein levels in a DDT/pyrethroid-resistant strain of Ae. aegypti. Recombinant ALDH9948 and ALDH14080 exhibited oxidase activities to catalyse the oxidation of a permethrin intermediate, phenoxybenzyl aldehyde (PBald), to phenoxybenzoic acid (PBacid). CONCLUSIONS/SIGNIFICANCE: ALDHs have been identified in association with permethrin resistance in Ae. aegypti. Characterisation of recombinant ALDHs confirmed the role of this protein in pyrethroid metabolism. Understanding the biochemical and molecular mechanisms of pyrethroid resistance provides information for improving vector control strategies.

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.

Wolbachia supergroups A and B in natural populations of medically important filth flies (diptera: muscidae, calliphoridae, and sarcophagidae) in Thailand.

Filth flies, belonging to suborder Brachycera (Family; Muscidae, Calliphoridae and Sarcophagidae), are a major cause of nuisance and able to transmit pathogens to humans and animals. These insects are distributed worldwide and their populations are increasing especially in sub-tropical and tropical areas. One strategy for controlling insects employs Wolbachia, which is a group of maternally inherited intracellular bacteria, found in many insect species. The bacteria can cause reproductive abnormalities in their hosts, such as cytoplasmic incompatibility, feminization, parthenogenesis, and male lethality. In this study we determined Wolbachia endosymbionts in natural population of medically important flies (42 females and 9 males) from several geographic regions of Thailand. Wolbachia supergroups A or B were detected in 7 of female flies using PCR specific for wsp. Sequence analysis of wsp showed variations between and within the Wolbachia supergroup. Phylogenetics demonstrated that wsp is able to diverge between Wolbachia supergroups A and B. These data should be useful in future Wolbachia-based programs of fly control.

A new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from Thailand, with keys to 11 species of the Simulium varicorne species-group.

Simulium (Gomphostilbia) piroonae sp. nov. is described on the basis of females, males, pupae, and mature larvae collected in Mae Hong Son Province, Thailand. This new species is placed in the chumpornense subgroup of the varicorne species-group in the subgenus Gomphostilbia by having the antenna with eight flagellomeres, the pleural membrane bare, the female subcosta lacking hairs, and the pupal gill basally divided into two somewhat inflated branches. It is distinguished from all 10 known species of this group by the pupal gill with six filaments. Keys to identify 11 species of the varicorne species-group are provided for females, males, pupae, and mature larvae.

Identification of salivary gland proteins depleted after blood feeding in the malaria vector Anopheles campestris-like mosquitoes (Diptera: Culicidae).

Malaria sporozoites must invade the salivary glands of mosquitoes for maturation before transmission to vertebrate hosts. The duration of the sporogonic cycle within the mosquitoes ranges from 10 to 21 days depending on the parasite species and temperature. During blood feeding salivary gland proteins are injected into the vertebrate host, along with malaria sporozoites in the case of an infected mosquito. To identify salivary gland proteins depleted after blood feeding of female Anopheles campestris-like, a potential malaria vector of Plasmodium vivax in Thailand, two-dimensional gel electrophoresis and nano-liquid chromatography-mass spectrometry techniques were used. Results showed that 19 major proteins were significantly depleted in three to four day-old mosquitoes fed on a first blood meal. For the mosquitoes fed the second blood meal on day 14 after the first blood meal, 14 major proteins were significantly decreased in amount. The significantly depleted proteins in both groups included apyrase, 5'-nucleotidase/apyrase, D7, D7-related 1, short form D7r1, gSG6, anti-platelet protein, serine/threonine-protein kinase rio3, putative sil1, cyclophilin A, hypothetical protein Phum_PHUM512530, AGAP007618-PA, and two non-significant hit proteins. To our knowledge, this study presents for the first time the salivary gland proteins that are involved in the second blood feeding on the day corresponding to the transmission period of the sporozoites to new mammalian hosts. This information serves as a basis for future work concerning the possible role of these proteins in the parasite transmission and the physiological processes that occur during the blood feeding.

A high resolution melting real time PCR for mapping of filaria infection in domestic cats living in brugian filariosis-endemic areas.

We present here a real time PCR with high resolution melting (HRM) analysis for determining the prevalence and distribution of filarial species in domestic cats residing in brugian filariosis endemic areas of Narathiwat province, Thailand. Filarial species can be clearly distinguished in a single well using a single pair of primers. Blood samples were taken from a total of 2039 domestic cats living in endemic areas. Microfilariae were detected in 5.7% of the sample, while the overall prevalence of filaria infection by HRM analysis was 6.6%. The filariae species found in the infected cats were Brugia malayi, Dirofilaria immitis, D. repens as well as Acanthocheilonema (Dipetalonema) reconditum. This is the first report of A. reconditum infection from Thailand. The study also observed an overlapping of the distribution areas of animal and human filariae. From a public health perspective, the distribution and prevalence of these nematodes warrant an appropriate drug-based prophylaxis to be administered to cats in the endemic areas to reduce the number of diseased carriers. Furthermore, this molecular approach is more sensitive than microfilariae detection, enables species identification and greatly facilitates the collection of epidemiological data. Thus, the present study may help to bridge human-animal interface by coordinating research outcomes with the control of zoonoses that is vitally important for human and veterinary public health.

Evidence to support natural hybridization between Anopheles sinensis and Anopheles kleini (Diptera: Culicidae): possibly a significant mechanism for gene introgression in sympatric populations.

BACKGROUND: Malaria caused by Plasmodium vivax is still a public health problem in the Republic of Korea (ROK), particularly regarding the recent re-emergence of this malarial species near the demilitarized zone in northwestern Paju City, Gyeonggi-do Province. Currently, at least 4 species (An. kleini, An. pullus, An. belenrae and An. lesteri) of the Hyrcanus Group are reported as possible natural vectors of vivax malaria in the ROK, and An. sinensis, which is the most dominant species, has long been incriminated as an important natural vector of this P. vivax. However, An. sinensis was ranked recently as a low potential vector. According to the discovery of natural hybrids between An. sinensis (a low potential vector for P. vivax) and An. kleini (a high potential vector for P. vivax) in Paju City, intensive investigation of this phenomenon is warranted under laboratory conditions. METHODS: Mosquitoes were collected during 2010-2012 from Paju City, ROK. Hybridization experiments used iso-female line colonies of these anophelines together with DNA analysis of ribosomal DNA [second internal transcribed spacer (ITS2)] and mitochondrial DNA [cytochrome c oxidase subunit I (COI)] of the parental colonies, F1-hybrids and repeated backcross progenies were performed intensively by using a PCR-based assay and pyrosequencing technology. RESULTS: The results from hybridization experiments and molecular investigations revealed that the mitochondrial COI gene was introgressed from An. sinensis into An. kleini. The An. sinensis progenies obtained from consecutive repeated backcrosses in both directions, i.e., F2-11 progeny [(An. sinensis x An. kleini) x An. sinensis] and F3-5 progeny [(An. kleini x An. sinensis) x An. kleini] provided good supportive evidence. CONCLUSIONS: This study revealed introgression of the mitochondrial COI gene between An. sinensis and An. kleini through consecutive repeated backcrosses under laboratory conditions. This new body of knowledge will be emphasized in reliable promising strategies in order to replace the population of An. kleini as a high potential vector for P. vivax, with that of a low potential vector, An. sinensis, through the mechanism of gene introgression in nature.

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.

MIDGUT ULTRASTRUCTURE OF FOURTH INSTAR OCHLEROTATUS TOGOI (DIPTERA: CULICIDAE).

The ultrastructure of the midgut of fourth instar Ochlerotatus togoi was investigated by light, scanning and transmission electron microscopy. This study was performed to provide information to help devise future control efforts aimed at the larval stages of this vector of filariasis. The fourth instar midgut was approximately 2 mm in length and consisted of three morphologically distinct cell types: epithelial, regenerative, and endocrine cells. There was a monolayer of epithelial cells on the luminal surface of the midgut, with multiple folds of the plasma membrane where it adjoined the basement membrane. Regenerative cells were scattered throughout the basal portion of the epithelium, along with endocrine cells. No evidence of division or differentiation was seen in any of the cell types. Six layers of the peritrophic matrix were observed in the gut lumen which separated ingested food from the midgut epithelial cells. Cytoplasmic protrusions were seen in many areas of the luminal midgut surface and numerous autophagosomes were seen in the epithelial cells of both early and late fourth instar larvae, suggesting autophagy is involved in the degeneration process of the midgut in preparation for pupation. This study provides a basis for understanding normal Oc. togoi larval midgut development. Further studies are needed to determine the factors that control larval growth and the nutritional state. Such information could be used to reduce adult fecundity and develop biological control mechanisms.

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.

Detection of the V1016G mutation in the voltage-gated sodium channel gene of Aedes aegypti (Diptera: Culicidae) by allele-specific PCR assay, and its distribution and effect on deltamethrin resistance in Thailand.

BACKGROUND: Resistance to pyrethroid insecticides is widespread among populations of Aedes aegypti, the main vector for the dengue virus. Several different point mutations within the voltage-gated sodium channel (VGSC) gene contribute to such resistance. A mutation at position 1016 in domain II, segment 6 of the VGSC gene in Ae. aegypti leads to a valine to glycine substitution (V1016G) that confers resistance to deltamethrin. METHODS: This study developed and utilized an allele-specific PCR (AS-PCR) assay that could be used to detect the V1016G mutation. The assay was validated against a number of sequenced DNA samples of known genotype and was determined to be in complete agreement. Larvae and pupae were collected from various localities throughout Thailand. Samples were reared to adulthood and their resistance status against deltamethrin was determined by standard WHO susceptibility bioassays. Deltamethrin-resistant and susceptible insects were then genotyped for the V1016G mutation. Additionally, some samples were genotyped for a second mutation at position 1534 in domain III (F1534C) which is also known to confer pyrethroid resistance. RESULTS: The bioassay results revealed an overall mortality of 77.6%. Homozygous 1016G individuals survived at higher rates than either heterozygous or wild-type (1016 V) mosquitoes. The 1016G mutation was significantly and positively associated with deltamethrin resistance and was widely distributed throughout Thailand. Interestingly, wild-type 1016 V mosquitoes tested were homozygous for the 1534C mutation, and all heterozygous mosquitoes were also heterozygous for 1534C. Mutant homozygous (G/G) mosquitoes expressed the wild-type (F/F) at position 1534. However, the presence of the 1534C mutation was not associated with deltamethrin resistance. CONCLUSIONS: Our bioassay results indicate that all populations sampled display some degree of resistance to deltamethrin. Homozygous 1016G mosquitoes were far likelier to survive such exposure. However, resistance in some populations cannot be explained due to kdr mutations and indicates that other resistance mechanisms are operating. The presence of this mutation alone does not fully explain the resistance phenotype we see among Thai Ae. aegypti populations.

Development of a facile system for mass production of Brugia malayi in a small-space laboratory.

Brugia malayi is one of the important lymphatic filarial nematodes that cause elephantiasis and disability in humans in the Asian region. Mass production at any stage of this nematode in both small laboratory animal hosts and mosquito vectors is still necessary in order to continue various research aspects. This study elucidated on the use of nonblood feeding or the autogenous Ochlerotatus togoi (Thailand strain) and male Mongolian jird (Meriones unguiculatus) system. This has brought about a low-cost and highly-effective procedure for the mass production of blood containing microfilariae, infective (L3) larvae, and adults of B. malayi under nonanimal-blood-feeding insectary and small-space animal-house conditions. The highly-infective rates (human-heparinized blood, 86.67-93.33; swine-heparinized blood, 83.33-96.67; bovine-heparinized blood, 76.67-80; chicken-heparinized blood, 73.33-76.67) and parasite loads (human-heparinized blood, 10.58-12.36; swine-heparinized blood, 8.40-10.38; bovine-heparinized blood, 9.75-9.91; chicken-heparinized blood, 3.41-4.65) of autogenous O. togoi to B. malayi and high numbers of adults recovered from ten B. malayi-infected male jirds (total = 327, 16-52) are good supportive evidence. In addition, all special techniques required for succeeding in the establishment of a facile system regarding these matters are detailed.

Simulium (Asiosimulium) furvum, a new species of black fly (Diptera: Simuliidae) from Thailand.

Simulium (Asiosimulium) furvum sp. nov. (Diptera: Simuliidae) is described from female, male, pupal, and larval specimens collected from Maewa National Park, Lampang Province, Thailand. This new species represents the fourth member of the subgenus Asiosimulium Takaoka & Chochoote, one of two small black fly subgenera endemic in the Oriental Region. It is characterized by a pear-shaped spermatheca in the female; a ventral plate in the male with a laterally compressed median keel directed ventrally and with a deep notch posteromedially, and aedeagal membrane with stout spines; and by 22 gill filaments in the pupa. Taxonomic notes are provided to separate this new species from three known species, Simulium (Asiosimulium) oblongum Takaoka & Choochote and Simulium (Asiosimulium) wanchaii Takaoka & Choochote, both from Thailand, and Simulium (Asiosimulium) suchitrae Takaoka from Nepal.

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.