Cytogenetic, crossing and molecular evidence of two cytological forms of Anopheles argyropus and three cytological forms of Anopheles pursati (Diptera: Culicidae) in Thailand

Nine and 11 isolines of Anopheles argyropus and Anopheles pursati, respectively, were established from individual females collected from cow-baited traps, and the characteristics of metaphase chromosomes were investigated in their F1-progenies. As determined by the different amounts of extra heterochromatin on sex chromosomes, 2 types of X (X1, X2) and Y (Y1,Y2), and 2 types of X (X1, X2) and 3 types of Y (Y1, Y2, Y3) chromosomes were obtained from An. argyropus and An. pursati, respectively. These types of sex chromosomes comprised 2 [Forms A (X1, Y1) and B (X1, X2, Y2)] and 3 [Forms A (X1, X2, Y1), B (X1, X2, Y2) and C (X2, Y3)] karyotypic forms of An. argyropus and An. pursati, respectively. All karyotypic forms acquired from An. pursati are new one that were discovered in this study, of which Forms A, B and C were found generally in Chiang Mai Province, while only 1 isoline of Form B was obtained in Ratchaburi Province. Form A was recovered from An. argyropus only in Ubon Ratchathani Province, whereas Form B from that species was found commonly in both Ubon Rathchathani and Nakhon Si Thammarat Provinces. Crossing experiments among the 2 and 3 isolines representing 2 and 3 karyotypic forms of An. argyropus and An. pursati, respectively, indicated genetic compatibility in yielding viable progenies and synaptic salivary gland polytene chromosomes through F2-generations. The conspecific natures of these karyotypic forms in both species were further supported by very low intraspecific sequence variations (average genetic distance: An. argyropus = 0.003-0.007, An. pursati = 0-0.005) of ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII).

Polygamy: the possibly significant behavior of Aedes aegypti and Aedes albopictus in relation to the efficient transmission of dengue virus.

The polygamous behavior of male Aedes aegypti (L.) and Ae. albopictus (Skuse) was investigated by co-habiting a newly-emerged male and females in a 30 cm3 cage (1 male: 20 females) for up to 5 consecutive days. As determined by insemination rates, the results indicated that one Ae. aegypti and Ae. albopictus male could successfully mate with 1.10 (0-4), 4.10 (1-8), 5.40 (4-8), 5.10 (2-8), 5.15 (3-9) and 0.20 (0-3), 1.70 (0-3), 2.35 (1-4), 2.30 (0-4), 2.35 (1-4) Ae. aegypti and Ae. albopictus females, respectively on day 1,2,3,4 and 5 consecutively. The possibly significant role of their polygamy in relation to dengue virus transmission is discussed.

Brugia malayi in a naturally infected cat from Narathiwat Province, southern Thailand.

Brugia malayi-like from an infected cat from Narathiwat Province, southern Thailand was identified intensively by microfilarial morphometry, acid phosphatase activity, and adult morphology. The results indicated that both microfilarial and adult characteristics conformed to the topotypic B. malayi.

Aedes albolateralis, a potential vector of nocturnally subperiodic Wuchereria bancrofti and dengue type 2 virus.

The susceptibility of Aedes albolateralis to nocturnally subperiodic Wuchereria bancrofti and dengue type 2 virus was investigated by using artificial membrane feeding and intrathoracic inoculation techniques, respectively. The results indicated that Ae. albolateralis was susceptible to nocturnally subperiodic W. bancrofti (susceptibility rate = 9.43%) and dengue type 2 virus (susceptibility rate = 100%), suggesting the potential vector of the two pathogens.

Development of Haplorchis taichui (Trematoda: Heterophyidae) in Mus musculus mice.

The development of Haplorchis taichui was studied in sixteen mice, Mus musculus. Metacercarial cysts of H. taichui were obtained from the freshwater fish, Thynnichthys thynnoides, collected in Chiang Mai Province, north Thailand. Approximately 200 active metacercariae were orally introduced into each mouse. Two mice were randomly sacrificed and necropsied daily from day 2-9 postinfection (pi). Two peaks of increment in the length and width of worms were found at day 3 and 7 pi. H. taichui was rapid in maturation, similar to other minute intestinal flukes. Rudimentary sex organs were found at day 2 pi. Spermatozoa in a seminal receptacle, vitellaria and eggs were seen as early as 3 days pi. The number of eggs increased daily to approximately 50-60 eggs thereafter. Mice can serve as a suitable experimental definitive host for harvesting adult H. taichui, especially in 1-week pi.

The application of ethanol-extracted Gloriosa superba for metaphase chromosome preparation in mosquitos.

The application of ethanol-extracted Gloriosa superba for metaphase chromosome preparation in adult and 4th larva Aedes aegypti revealed that 0.5-8% ethanol-extracted Gl. superba solution could be used instead of 1% colchicine in Hanks' balanced salt solution. For adult mosquitos, the metaphase rates and average number of metaphase chromosomes per positive mosquito after intrathoracic inoculation with 1-2% ethanol-extracted Gl. superba solution were 100% and 11.8 (2-16) -12.6 (3-28) in females, and 80-90% and 16.5 (1-52) - 29.89 (1-72) in males, whereas the inoculation with 1% colchicine solution yielded 80% and 50% metaphase rates, and 18.25 (1-40) and 16.5 (2-53) average number of metaphase chromosomes per positive mosquito in females and males, respectively. For 4th stage larvae, the metaphase rates and average number of metaphase chromosomes per positive mosquito after incubation with 0.5-8% ethanol-extracted Gl. superba solution were 90-100% and 14.42 (1-65) - 64 (19-137), while incubation with 1% colchicine solution yield 100% metaphase rate and 10.9 (7-15) average number of metaphase chromosomes per positive mosquito.

Comparative morphometry, morphology of egg and adult surface topography under light and scanning electron microscopies, and metaphase karyotype among three size-races of Fasciola gigantica in Thailand.

Comparative morphometry of eggs and adults under light microscope, and morphology of adults under scanning electron microscope (SEM) were undertaken in the three size-races (< 25 mm, 25-35 mm, > 35 mm) of Fasciola gigantica (Thailand strain). Morphometric examination revealed intraspecific variation with respects to the dimensions of eggs and adults, whereas surface topography of the three size-race adults under SEM was morphologically similar. The observations on mitotic metaphase chromosomes of spermatogonial cells from testes of the three size-races revealed 2n=20 (diploid type), and no karyotypic difference was observed among them. The meiotic metaphase chromosome was 10 bivalents in primary spermatocyte in diplotene to diakinesis, and many mature spermatozoa were seen in the testicular preparations.

Autogenous Aedes togoi sub-colony (Chanthaburi, Thailand strain), an efficient laboratory vector in study of filariasis.

Comparative filarial susceptibility and biology between stock colony and selectively autogenous Aedes. togoi sub-colony were carried out to determine the laboratorial vector-capacity and viability of autogenous sub-colony. The results of susceptibility revealed that the selectively autogenous Ae. togoi sub-colony yielded higher susceptibility than the stock colony, ie Dirofilaria immitis: susceptibility rates=80.00% [Exp1(F8)] and 76.19% [Exp2(F17)] (autogenous sub-colony), 53.33% (Exp1) and 71.43% (Exp2) (stock colony); Brugia malayi: susceptibility rates=83.33% [Exp1(F17)] and 84.38% [Exp2(F19)] (autogenous sub-colony), 81.25% (Exp1) and 75.00% (Exp2) (stock colony), but not at the level of statistically significant differences except the Exp1 of D. immitis, which was significant difference. In addition, the average No. L3 per infected mosquito in the selective autogenous sub-colony (D. immitis: Exp1=3.37, Exp2=3.19; B. mallayi: Exp1=8.80, Exp2=3.37) was also higher than in stock colony (D. immitis: Exp1=2.44, Exp2=2.73; B. malayi: Exp1=7.85, Exp2=3.02), but not at the level of statistically significant difference. The results of comparisons on some biological aspects demonstrated that most of the cases they have similar biology except the average egg deposition per gravid female of stock colony (130.17+/-43.33) was significantly more than selectively autogenous sub-colony(F9) (94.33+/-13.69), egg length x width 575.62+/-18.06 microm x 186.15+/-9.35 microm of stock colony was significantly larger than selectively autogenous sub-colony(F9) 560.49+/-18.96 microm x 177.99+/-8.40 microm, and mean longevity of adult female of stock colony [41.60(6-61)] was significantly longer than selectively autogenous sub-colony(F9) [35.00(5-39)]. The selectively autogenous sub-colony was established and twenty-two successive generations have been colonized.

Larvicidal, adulticidal and repellent effects of Kaempferia galanga.

Four fractions of Kaempferia galanga (hexane fraction, dichloromethane fraction 1, dichloromethane fraction 2 and methanolic fraction) were tested for larvicidal activity toward fourth instar Culex quinquefasciatus. The hexane fraction was found to exhibit the highest larvicidal effect with the LC50 of 42.33 ppm. Testing for adulticidal activity, the hexane fraction did not show any promising adulticidal effect. However, it caused a knockdown effect which might be useful as a repellent. It was then tested for repellent activity in human volunteers both in laboratory and field studies. In a laboratory study, the hexane fraction possessed repellency against Aedes aegypti (ED50 value of 30.73 microg/cm2), and provided biting protection for 3 hours. In a field study, it could protect against certain mosquitos, ie, Armigeres subalbatus, Anopheles barbirostris, An. aconitus, Mansonia uniformis, Cx. quinquefasciatus, Cx. gelidus, Cx. tritaeniorhynchus and Ae. aegypti. The hexane fraction did not cause dermal irritation when applied on human skin.

Isoenzyme study and hybridization of two forms of Anopheles sinensis (Diptera: Culicidae) in northern Thailand.

The screening of ten isoenzymes of two forms of Anopheles sinensis, Form A and B, using electrophoretic gels revealed that Est-5(96) allele was the marker in both 4th larva and adult female of An. sinensis Form B, whereas it was lacking in Form A. Hybridization tests of the two sinensis forms were done by induced copulation. The results of crosses indicated that they were genetically compatible, providing viable progeny and completely synaptic polytene chromosomes.

Screening for larvicidal activity of ten carminative plants.

Ten species of plants, reported to possess carminative property, were screened for larvicidal potential against Culex quinquefasciatus by exposing early 4th instar larvae to a series of concentrations of the ethanolic extracts of the plants. Mortality counts were made after 24 hours exposure. Probit analysis using computerized Harvard Programming (Hg1, 2) was employed to determine the LC50, LC95 and LC99 values in order to compare the larvicidal potency of the ten plants. Marked larvicidal effects were seen with Kaempferia galanga, Illicium vernum and Spilanthes acmella having LC50 values of 50.54, 54.11 and 61.43 ppm, respectively.

Comparative susceptibility of two forms of Anopheles sinensis Wiedemann 1828 (Diptera : Culicidae) to infection with Plasmodium falciparum, P. vivax, P. yoelii and the determination of misleading factor for sporozoite identification.

Two karyotypic forms of laboratory-raised Anopheles sinensis, ie Form A (XY1) and Form B (XY2), were experimentally infected with various indigenous strains of Plasmodium falciparum and P. vivax using an artificial membrane feeding technique, and a rodent malaria, P. yoelii, using a direct feeding technic and dissected 7-9 days and 10-15 days after feeding for oocyst and sporozoite rates, respectively. The results revealed that two forms of An. sinensis were refractory vectors for P. falciparum and P. yoelii since 0% of oocyst and sporozoite rates were obtained, but poor vectors for P. vivax since 0.00-85.71% and 0.00-5.88% of oocyst and sporozoite rates were recovered. The sporozoite-like crystal found in the median lobe of the salivary gland of An. sinensis which could be a misleading factor in identification of true sporozoites in the salivary glands is reported for the first time.

Paragonimiasis in Mae Hong Son Province northern Thailand: case report.

Two cases of paragonimiasis have been reported from Srisangval Hospital, Mae Hong Son Province, Thailand. These two patients (Thai-Karen) resided in Nam-Piang-Din, Mae Hong Son. The preliminary survey for epidemiological data in Nam-Piang-Din, Mae Hong Son revealed that most of Thai-Karen villagers had the habit of eating raw crabs and fresh water prawns. A survey for intermediate and reservoir hosts are still in progress and this area can be considered as an additional area for paragonimiasis in Thailand.

Bancroftian filariasis in Tambol Na-Sai, Amphoe Li, Lamphun Province: a preliminary survey for microfilaraemia.

A preliminary survey for Bancroftian filariasis in villagers of Ban Prabat Heuy Tom, Tambol Na-Sai, Amphoe Li, Lamphun Province was performed between July 12, 1986 and March 19, 1987. A total of 1,435 villagers comprising 668 females and 767 males were examined for microfilaraemia in peripheral blood. Microfilariae were found in five males of which two cases were found to have developed hydrocele at a lesser degree and all of them had formerly resided in an endemic area of Tak Province. Microfilarial periodicity was studied in two cases, and it was shown to be nocturnally subperiodic.

Studies on the Japanese encephalitis vectors in Amphoe Muang, Chiang Mai, Northern Thailand.

From February 1987 to January 1988, biological and ecological studies were made to obtain the basic knowledge of Japanese encephalitis vectors, Culex tritaeniorhynchus, Cx. gelidus, and Cx. fuscocephala, in Amphoe Muang, Chiang Mai, northern Thailand. The following results were found. Peaks in the population densities of the vectors as measured four times a month, by UV-light trap and human-baited trap collections, occurred during rainy season. The JE vectors in rural areas showed a sharp rise in the population in July when most of the rice fields were ploughed and a marked decline in mosquito population densities occurred after transplanting in August when the fields were flooded. The average number of larvae plus pupae per m2 in rice fields was highest in July when the fields were ploughed, but in the period from transplanting to harvesting (August to November), the densities were very low. Daily survival rates of the adult females, as estimated from parous rates, were mostly as high as 0.7 throughout the year.

Alteration in Aedes togoi susceptibility to Brugia pahangi microfilariae induced by Aedes albopictus thoracic homogenate.

Refractoriness to Brugia pahangi microfilarial infection could be induced in the normally susceptible Aedes togoi mosquitoes by intrathoracic injection with crude thoracic homogenate of the refractory Aedes albopictus mosquitoes. The crude thoracic homogenate contained three Sephadex G-200 protein profiles of which the first profile showed strongest inhibition to the parasite development.