Comparative identification of protein profiles and major allergens of saliva, salivary gland and whole body extracts of mosquito species in Thailand.

Allergic reactions to mosquito bites, such as generalized urticaria or severe local reactions are common problems worldwide. The diverse sources of allergen prepared from different mosquito body parts usage are a major obstacle to obtaining safe and effective tests and immunotherapy for mosquito bite allergy. Thus, the reactions are often not recognized and allergen immunotherapy is seldom used for severe reaction to mosquito bites. In a search for appropriate allergen sources, the protein profiles of saliva, salivary glands and whole body extracts were comparatively analyzed from 4 common mosquito species of Thailand and/or South East Asia; viz. Culex quinquefasciatus, Aedes aegypti, Aedes albopictus and a zoophilic strain, Anopheles minimus. The major allergens in the extracts which elicited specific IgE responses in the pooled sera of subjects allergic to mosquito bites were identified. It was concluded that mosquito saliva was the best source of allergens. Additionally, both species-specific and species-shared allergens of the 4 mosquito species were identified. The major saliva allergens having MWs of 36, 32 and 22 kDa were identified. The identificstion of major allergens should facilitate the production of specific recombinant allergens and contribute to improvement in the diagnosis and specific immunotherapy of Thai mosquito bite allergy patients.

Effect of crude extract of Solanum xanthocarpum against snails and mosquito larvae.

The ethanolic crude extract from Solanum xanthocarpum was investigated for its molluscicidal activity against Biomphalaria glabrata, the snail vector of Schistosoma mansoni, and Indoplanorbis exustus, the snail vector of intestinal echinostomiasis and Schistosoma spindale, together with the larvicidal activity against the larvae of Aedes aegypti, mosquito vector of dengue hemorrhagic fever and Culex quinquefasciatus, the mosquito vector of urban bancroftian filariasis. The bioassays were carried out following the methods recommended by the World Health Organization. For molluscicidal activity, the LC50 against Bi. glabrata and I. exustus were reported at 163.85 and 198.00 mg/l while the LC90 were 219.33 and 236.80 mg/l, respectively. Regarding mosquito larvicidal activity, the LC50 against the larvae of Ae. aegypti and Cx. quinquefasciatus were 788.10 and 573.20 mg/l, while the LC90 were 1288.91 and 1066.93 mg/l, respectively. These results suggest a preparation of ingredients from this plant may be used as a biological larvicide for these vectors in the field.

Control of mosquito vectors of tropical infectious diseases: (3) susceptibility of Aedes aegypti to pyrethroid and mosquito coils.

We collected 11 groups of Aedes aegypti mosquitoes from various locations of Thailand. After rearing in the laboratory, the colonies were tested for KT50 values to dl,d-T80-allethrin 0.5% mosquito coils in a 25 m3 room semi-field test and KD50 and LD50 values to dl,d-T80-allethrin by a topical application method. Two groups of mosquitoes were susceptible to allethrin similar to a SS (known allethrin sensitive) group, and other 9 groups showed various levels of lower susceptibility to allethrin; of these 6 had susceptibilities similar to a BS (known allethrin resistant) group with extremely low susceptibility, while the remaining 3 groups had susceptibilities to allethrin between the SS and BS groups. The KD50 values with the topical application were found to correlate highly with the KT50 values in the 25 m3 room semi-field test, providing a useful test method for insect susceptibility evaluation. The allethrin mosquito coils, even at higher concentrations, had no activity against the 6 decreased susceptibility groups, similar to the BS group. With the 25 m3 room semi-field test, mosquito coils with d,d-T-prallethrin at concentrations of 0.1 to 0.15% plus a synergist and those with methoxymethyl-tetrafluorobenzyl tetramethylcyclopropane carboxylate (K-3050) at a concentration of 0.1% plus a synergist were found to be highly effective against these mosquito groups. These two pyrethroids had smaller KD50 and LD50 values for topical application, and were more effective than dl,d-T80-allethrin, having the potential to control Ae. aegypti mosquitoes with low allethrin susceptibility.

Adherence to antimalarial drug therapy among vivax malaria patients in northern Thailand.

Vivax malaria is a significant cause of morbidity due to malaria in northern Thailand, accounting for approximately 50% of all malaria cases. The objective of this study was to determine the behavioural factors associated with adherence to the standard 14-day course of chloroquine and primaquine, prescribed from malaria clinics, among patients with vivax malaria. A retrospective study was conducted among 206 patients living in Muang and Mae Sa Riang districts of Mae Hon Son province in northern Thailand. Data on adherence and potential behavioural factors relating to adherence were collected using a structured interviewer-administered questionnaire and supplemented with qualitative data from focus-group interviews. The results indicated that 76.21% of the 206 patients with vivax malaria did not complete the medication course. The adherence of the patients was associated with knowledge scores of malaria (adjusted odds ratio [AOR]=2.2, 95% confidence interval [CI] 1.1-4.5) and accessing drug prescription scores (AOR=5.6, 95% CI 2.13-15.3). Therefore, further effort is needed to educate patients with vivax malaria on knowledge of malaria and its treatment with simple health messages and encourage them to adhere to their treatment.

Control of mosquito vectors of tropical infectious diseases: (1) bioefficacy of mosquito coils containing several pyrethroids and a synergist.

The bioefficacy of mosquito coils containing several pyrethroids were tested in a 25 m3 room against Culex pipiens quinquefasciatus, Aedes aegypti and Anopheles dirus. The test results were compared with tests against Culex pipiens pallens in Japan. Based on the KT50 values (the 50% knockdown time) of mosquito coils containing dl, d-T80-allethrin, d, d-T-prallethrin and methoxymethyl-tetrafluorobenzyl tetramethyl-cyclopropanecarboxylate (K-3050) at doses of 0.05-0.5% (w/w) with or without a synergist, the pyrethroid susceptibility of the four mosquito species was as follows: Cx. p. quinquefasciatus was several times more tolerant to pyrethroids than Cx. p. pallens, Ae. aegypti was a further several times more tolerant than Cx. p. quinquefasciatus, and An. dirus was more susceptible than Cx. p. pallens (KT50 value: about half of Cx. p. pallens). The order of their susceptibilities is common for pyrethroids. Mosquito coils containing d, d-T-prallethrin and K-3050 at doses of 0.05-0.2% (w/w) and N-(2-ethylhexyl)bicycle-[2,2,1]-hept-5-ene-2,3-dicarboxyimide as a synergist at a ratio of 2 times the active ingredient were highly effective against Ae. aegypti, the most important mosquito vector for dengue fever.

Control of mosquito vectors of tropical infectious diseases: (2) pyrethroid susceptibility of Aedes aegypti ( l) collected from different sites in Thailand.

Four strains (SS, BS, A and B) of Aedes aegypti collected from different sites in Bangkok and at different times were examined for their pyrethroid susceptibility. Mosquito coils containing dl, d-T80-allethrin, d, d-T-prallethrin and methoxymethyl-tetrafluorobenzyl tetramethyl-cyclopropanecarboxylate (K-3050) with or without a synergist were tested by the 25 m3 semi-field test method. One strain (SS) was the most susceptible with KT50 values of about < 30 minutes for all mosquito coils, while the other three strains (BS, A and B) were found to be around 10 to 20 times more tolerant to pyrethroids than the SS strain. A similar tendency for the pyrethroid susceptibility of the four strains was obtained with tests by topical application method. In field efficacy tests, mosquito coils with d, d-T-prallethrin 0.20% plus N-(2-ethylhexyl)bicycle-[2,2,1]- hept-5- ene-2,3-dicarboxyimide as a synergist exhibited a repellent effect of about 85%, while those with K-3050 0.10% plus the synergist exhibited a greater repellent effect of about 90%. In contrast, the repellent effect of commercial dl, d-T80-allethrin 0.20% coils was as low as about 50%. The d, d-T-prallethrin and K-3050 coils with the synergist were confirmed to be highly effective in repelling Ae. aegypti.

The geometry of the wing of Aedes (Stegomyia) aegypti in isofemale lines through successive generations.

Under a common laboratory environment, three isofemale lines of Aedes (Stegomyia) aegypti were used to score metric properties (size and shape) of the wings during 10 generations. Since the number of generations was much higher than the number of founders in each line, genetic drift was expected to occur. Size tended to slightly increase with time, but its variation among the successive generations did not show any detectable information specific to each line. Shape could discriminate among lines in females. Males of lines A and B were not discriminated before generation 8, after which they became completely separated. For each line at each generation, the variance of size and the metric disparity index (as an estimate of shape variance) were higher in females than in males. From one generation to another, the within line shape variance decreased in both sexes, while shape similarity progressively and consistently decreased between males of lines A and B. At each generation, in both sexes, shape variance of the pooled lines was higher than that of each line separately, a pattern not observed for size variance. In conclusion, shape, as a metric property, was closer to a genetic character than size: (i) it showed modifications compatible with the hypothesis of genetic drift and (ii) its variation was related to the complexity of the sample.

An empowerment program to enhance women's ability to prevent and control malaria in the community, Chiang Mai Province, Thailand.

Paulo Freire's theory was modified to empower a women's group in Chiang Mai Province, Thailand, to prevent and control malaria. This study conducted an intervention in Mueang Na Wan Village, Mueang Na Sub-district, Chiang Dao District, Chiang Mai Province, where 45 women were systematically recruited into the study cohort. Navail Village was selected as a control village because it resembled the intervention village. The empowerment program emphasized enhancement of malaria preventive levels, using insecticide-treated bed nets, self-esteem, and self confidence expectation to prevent and control malaria. Intensive training was conducted and activities performed among the women's group, with 10 participatory meetings in all. Data collection was conducted for the pre-test in month 1, and post-intervention in months 3, 6, 9, and 12. The qualitative methods used were focus-group discussions, non-participant observations, and in-depth interviews with housewives, their husbands, and youths at risk for malaria. The results showed that, post-intervention, there were significantly increased levels for malaria preventive behaviors, behaviors of using insecticide-treated nets, self-esteem, and self confidence expectations, in the intervention village compared with the control village. Insecticide-treated net usage and insecticide-treated net usage behaviors increased in the intervention village more than before and more than that in the control village. The women's group in the intervention village created the following plans, which were crucial to malaria prevention: (1) a family protection plan, (2) providing malaria education to community members, (3) a mosquito-control campaign, (4) scaling-up insecticide-impregnated bed nets, and (5) malaria control among foreign laborers. Finally, the empowered women's group performed sustainable activities. Between malaria-prevention activities, they conducted a joint program to raise income for their families.

Laboratory colonization of Mansonia mosquitoes with an emphasis on Ma. annulata and Ma. bonneae.

The present study records the first successful colonization of Mansonia annulata and describes colony maintenance with modification of rearing medium and host plants. Three species of Mansonia mosquitoes (Ma. uniformis, Ma. indiana and Ma annulifera) were successfully reared in ambient environments with adult emergence rates > 50%, while Ma. bonneae and Ma. dives yielded emergence rates > 30%. Colonization of Ma. annulata was modified and improved so that they were successfully raised to adult with emergence rates of 23%. Tube sedge, Lepironia articulata, was utilized as a host plant and peat swamp water was used as a rearing medium. Yeast and small lizard droppings were added daily to the larval medium to maintain microorganisms and pH in the infusion. However, identifying suitable culture medium remains an obstacle to establishing colonies of Ma. annulata, as the culture medium is difficult to mimic in the laboratory. Further study, focusing particularly on larval attachment substrates and rearing medium, is needed to develop a standardized and practical rearing technique for Mansonia mosquitoes.

Deployment of early diagnosis and mefloquine-artesunate treatment of falciparum malaria in Thailand: the Tak Malaria Initiative.

BACKGROUND: Early diagnosis and treatment with artesunate-mefloquine combination therapy (MAS) have reduced the transmission of falciparum malaria dramatically and halted the progression of mefloquine resistance in camps for displaced persons along the Thai-Burmese border, an area of low and seasonal transmission of multidrug-resistant Plasmodium falciparum. We extended the same combination drug strategy to all other communities (estimated population 450,000) living in five border districts of Tak province in northwestern Thailand. METHODS AND FINDINGS: Existing health structures were reinforced. Village volunteers were trained to use rapid diagnostic tests and to treat positive cases with MAS. Cases of malaria, hospitalizations, and malaria-related deaths were recorded in the 6 y before, during, and after the Tak Malaria Initiative (TMI) intervention. Cross-sectional surveys were conducted before and during the TMI period. P. falciparum malaria cases fell by 34% (95% confidence interval [CI], 33.5-34.4) and hospitalisations for falciparum malaria fell by 39% (95% CI, 37.0-39.9) during the TMI period, while hospitalisations for P. vivax malaria remained constant. There were 32 deaths attributed to malaria during, and 22 after the TMI, a 51.5% (95% CI, 39.0-63.9) reduction compared to the average of the previous 3 y. Cross-sectional surveys indicated that P. vivax had become the predominant species in Thai villages, but not in populations living on the Myanmar side of the border. In the displaced persons population, where the original deployment took place 7 y before the TMI, the transmission of P. falciparum continued to be suppressed, the incidence of falciparum malaria remained low, and the in vivo efficacy of the 3-d MAS remained high. CONCLUSIONS: In the remote malarious north western border area of Thailand, the early detection of malaria by trained village volunteers, using rapid diagnostic tests and treatment with mefloquine-artesunate was feasible and reduced the morbidity and mortality of multidrug-resistant P. falciparum.

Meteorological, environmental remote sensing and neural network analysis of the epidemiology of malaria transmission in Thailand.

In many malarious regions malaria transmission roughly coincides with rainy seasons, which provide for more abundant larval habitats. In addition to precipitation, other meteorological and environmental factors may also influence malaria transmission. These factors can be remotely sensed using earth observing environmental satellites and estimated with seasonal climate forecasts. The use of remote sensing usage as an early warning tool for malaria epidemics have been broadly studied in recent years, especially for Africa, where the majority of the world's malaria occurs. Although the Greater Mekong Subregion (GMS), which includes Thailand and the surrounding countries, is an epicenter of multidrug resistant falciparum malaria, the meteorological and environmental factors affecting malaria transmissions in the GMS have not been examined in detail. In this study, the parasitological data used consisted of the monthly malaria epidemiology data at the provincial level compiled by the Thai Ministry of Public Health. Precipitation, temperature, relative humidity, and vegetation index obtained from both climate time series and satellite measurements were used as independent variables to model malaria. We used neural network methods, an artificial-intelligence technique, to model the dependency of malaria transmission on these variables. The average training accuracy of the neural network analysis for three provinces (Kanchanaburi, Mae Hong Son, and Tak) which are among the provinces most endemic for malaria, is 72.8% and the average testing accuracy is 62.9% based on the 1994-1999 data. A more complex neural network architecture resulted in higher training accuracy but also lower testing accuracy. Taking into account of the uncertainty regarding reported malaria cases, we divided the malaria cases into bands (classes) to compute training accuracy. Using the same neural network architecture on the 19 most endemic provinces for years 1994 to 2000, the mean training accuracy weighted by provincial malaria cases was 73%. Prediction of malaria cases for 2001 using neural networks trained for 1994-2000 gave a weighted accuracy of 53%. Because there was a significant decrease (31%) in the number of malaria cases in the 19 provinces from 2000 to 2001, the networks overestimated malaria transmissions. The decrease in transmission was not due to climatic or environmental changes. Thailand is a country with long borders. Migrant populations from the neighboring countries enlarge the human malaria reservoir because these populations have more limited access to health care. This issue also confounds the complexity of modeling malaria based on meteorological and environmental variables alone. In spite of the relatively low resolution of the data and the impact of migrant populations, we have uncovered a reasonably clear dependency of malaria on meteorological and environmental remote sensing variables. When other contextual determinants do not vary significantly, using neural network analysis along with remote sensing variables to predict malaria endemicity should be feasible.

Insecticide susceptibility of mosquitoes invading tsunami-affected areas of Thailand.

In order to control the mosquitoes invading tsunami-affected areas of Thailand, the insecticide susceptibility status of field larvae and mosquitoes (Anopheles sundaicus and Culex sitiens) was tested under laboratory conditions. Larval bioassay tests were conducted using the WHO standard method. Three larvicides: temephos, malathion, and plant extract (ethanolic extract of the Southeast Asian long pepper. Piper retrofractum Vahl), were used in the experiments. The results revealed that Cx. sitiens was more susceptible to temephos than malathion and the plant extract, with LC50 ranges of 0.0008-0.0014 mg/l, 0.0046-0.0078 mg/l, and 5.3180-10.1030 mg/l, respectively. Cx. quinquefasciatus showed greater tolerance to every tested larvicide than Cr sitiens. Adult bioassay tests using a WHO test kit and diagnostic doses of 5% malathion, 0.75% permethrin, 0.05% deltamethrin, and 4% DDT were also conducted. The results revealed that Cx. sitiens and An. sundaicus were susceptible to all tested insecticides. The LT50 of 5% malathion ranged between 25.7-26.0 minutes for Cx. sitiens, and 44.7 minutes for An. sundaicus. In addition, Cx. quinquefasciatus showed susceptibility to malathion, with LT10 of 19.7 minutes. However, it showed resistance to both pyrethroid insecticides, with LT50 of 33.1 minutes for 0.75% permethrin, and 19.6 minutes. for 0.05% deltamethrin; it showed low percentage mortality at 24 hour post-exposure, of 48 and 32%, respectively. In conclusion, every tested larvicide could be used for controlling Cx. sitiens larvae, even in brackish water, pyrethroid insecticides for adult Cx. sitiens and An. sundaicus, and malathion for all three species.

Determination of lead toxicity in Culex quinquefasciatus mosquitoes in the laboratory.

Laboratory investigations were carried out to study the effects of lead toxicity and lead uptake on Culex quinquefasciatus mosquitoes. Three different concentrations of lead nitrate were used in laboratory tests (0.05, 0.1, and 0.2 mg/l). An atomic absorption spectrometer (AAS) was used to the determine lead concentrations. The results showed that lead significantly reduced hatching, egg-production, and emergence rates, compared with the unexposed group (p < 0.05). The ratio of female to male offspring was 3.64:1, which was observed in the second generation, after the parents were exposed to 0.2 mg/l lead. No effects were observed on oviposition preference, larval weight, or larval deformation. The LC50 of lead against Cx. quinquefasciatus larvae within 24 hours was 0.18 mg/l. There was a significant increase in lead uptake related to increased lead exposure in mosquito larvae (p < 0.05). The bioconcentration factor (BCF) showed that the lead concentration in the larvae was 62 times greater than in the water. The lead concentration from parents to offspring reduced in the first and second generations (p < 0.05). There was no significant difference between female and male mosquitoes in lead concentration (p > 0.05).

First isolation of Japanese encephalitis from Culex quinquefasciatus in Thailand.

Isolation of Japanese encephalitis (JE) virus using C6/36 cell and immunofluorescence virus antigen detection techniques was attempted from female mosquitoes collected with CDC gravid traps in Samut Songkhram Province in the central region and in Phuket Province in southern Thailand, in 2003. One thousand and eighty female mosquitoes including 6 species of the Culicidae family (Culex quinquefasciatus, Cx. gelidus, Cx. tritaeniorhynchus, Cx. whitmorei, Cx. vishnui complex, Cx. s.g. culiciomyia) (pooled by specific specimen), were processed for virus isolation. Two pools of Cx. quinquefasciatus yielded a JE virus isolation. This represents the first report of JE virus isolation from Cx. quinquefasciatus in Thailand.

Comparative studies on the biology and filarial susceptibility of selected blood-feeding and autogenous Aedes togoi sub-colonies.

Blood-feeding and autogenous sub-colonies were selected from a laboratory, stock colony of Aedes togoi, which was originally collected from Koh Nom Sao, Chanthaburi province, Southeast Thailand. Comparative biology and filarial susceptibility between the two sub-colonies (blood-feeding: F11, F13; autogeny: F38, F40) were investigated to evaluate their viability and vectorial capacity. The results of comparison on biology revealed intraspecific differences, i.e., the average egg deposition/gravid female (F11/F38; F13/F40), embryonation rate (F13/F40), hatchability rate (F11/F38; F13/F40), egg width (F11/F38), wing length of females (F13/F40), and wing length and width of males (F11/F38) in the blood-feeding sub-colony were significantly greater than that in the autogenous sub-colony; and egg length (F11/F38) and width (F13/F40), and mean longevity of adult females (F11/F38) and males (F13/F40) in the blood-feeding sub-colony were significantly less than that in the autogenous sub-colony. The results of comparison on filarial susceptibility demonstrated that both sub-colonies yielded similar susceptibilities to Brugia malayi [blood-feeding/autogeny = 56.7% (F11)/53.3%(F38), 60%(F13)/83.3%(F40)] and Dirofilaria immitis [blood-feeding/autogeny = 85.7%(F11)/75%(F38), 45%(F13)/29.4%(F40)], suggesting autogenous Ae. togoi sub-colony was an efficient laboratory vector in study of filariasis.

Comparative studies on the biology and filarial susceptibility of selected blood-feeding and autogenous Aedes togoi sub-colonies

Blood-feeding and autogenous sub-colonies were selected from a laboratory, stock colony of Aedes togoi, which was originally collected from Koh Nom Sao, Chanthaburi province, Southeast Thailand. Comparative biology and filarial susceptibility between the two sub-colonies (blood-feeding: F11, F13; autogeny: F38, F40) were investigated to evaluate their viability and vectorial capacity. The results of comparison on biology revealed intraspecific differences, i.e., the average egg deposition/gravid female (F11/F38; F13/F40), embryonation rate (F13/F40), hatchability rate (F11/F38; F13/F40), egg width (F11/F38), wing length of females (F13/F40), and wing length and width of males (F11/F38) in the blood-feeding sub-colony were significantly greater than that in the autogenous sub-colony; and egg length (F11/F38) and width (F13/F40), and mean longevity of adult females (F11/F38) and males (F13/F40) in the blood-feeding sub-colony were significantly less than that in the autogenous sub-colony. The results of comparison on filarial susceptibility demonstrated that both sub-colonies yielded similar susceptibilities to Brugia malayi [blood-feeding/autogeny = 56.7 percent (F11)/53.3 percent(F38), 60 percent(F13)/83.3 percent(F40)] and Dirofilaria immitis [blood-feeding/autogeny = 85.7 percent(F11)/75 percent(F38), 45 percent(F13)/29.4 percent(F40)], suggesting autogenous Ae. togoi sub-colony was an efficient laboratory vector in study of filariasis

Malaria and enterobiasis among Karen Long-neck tribe in Mae Hong Son Province.

In Thailand, Mae Hong Son Province is highly endemic for malaria. Knowing this, the local Health Department has introduced a program to educate local residents about the risk factors, in particular the dangers and symptoms of malaria. This study was conducted to evaluate these efforts, by determining the number of malaria infections in a segment of the population, and also by testing for enterobiasis among a group of its children. Two villages in Mae Hong Son Province were chosen for this purpose with a combined population of about 300. Of these, 195 were screened for malaria. Two subjects were diagnosed positive for malaria by microscopy. One of these two villages was chosen to screen for Enterobius vermicularis infection in children as well. Out of 69 stool samples, five (7%) showed infection with E. vermicularis: three with a low number of eggs (1-50), and two with a high number of eggs (>100). Compared with infection rates in similar studies, the results of this study indicate that the Health Department's efforts are meeting with relative success. The low prevalence of infection indicates that the villagers are using the information they have received to help combat infection.

Parasitic infections among Karen in Kanchanaburi Province, western Thailand.

The purpose of this study was to determine the rate of intestinal parasitic and malarial infections during a period of low infection among the residents of remote Karen villages in Thailand. Fifty-five males and 64 females, aged 6 months to 70 years, were examined for malaria by thick blood smears using the Giemsa staining technique. Of the 119 subjects, 4 (3.36%) showed positive for malaria with vivax gametocytes. Results suggested that mass screening was not an effective way for diagnosing malaria. Stool samples were examined under a light microscope. The overall intestinal parasitic infection rates were 38.24% in 34 males, and 36.11% in 36 females. These were hookworm (17.14%), Ascaris lumbricoides (7.14%), Trichuris trichiura (1.43%), Strongyloides stercoralis (7.14%), Taenia spp (1.43%), Entamoeba histolytica (1.43%), Entamaeba coli (10.00%) and Giatdia lamblia (1.43%). The highest (55.55%) and lowest (16.66%) rates of infection were observed in age groups 0-5 and over 45, years respectively. In addition, A. lumbricoides and T. trichiura were found more frequently in children, while hookworms was found similarly in every age group. Results showed that the Karen living along the western border of Thailand possessed high rates of intestinal parasitic infections. Strict monitoring and control programs for these parasites should be implemented.

Intraspecific hybridization of Anopheles minimus (Diptera: Culicidae) species A and C in Thailand.

Hybridization tests of laboratory-raised, isolines of Anopheles minimus, species A and C were conducted by induced copulation. The three isolines were established based on three morphological variants of wild-caught, fully engorged females and two distinct types of metaphase chromosomes. They were An. minimus species A: V form (X1,Y1), M form (X2,Y1); species C: P form (X3,Y2). The results of reciprocal and back crosses indicated that the two morphologically variant forms of species A were genetically compatible, providing viable progeny and completely synaptic salivary gland polytene chromosomes, whereas they were genetically incompatible with species C and/or the P form. Hybrid progeny was only obtained from both forms of species A females x species C males, but asynaptic salivary gland polytene chromosomes on 3L and partial development of ovarian follicles in females were seen. Back crosses of F1 hybrid males with parental species A females provided viable progeny, while back crosses of F1 hybrid females with parental species C males provided progeny of low viability and adult males with abnormal spermatozoa, suggesting the partial reproductive isolation of An. minimus species A and C.

Intraspecific hybridization of two karyotypic forms of Anopheles vagus (Diptera: Culicidae) and the related egg surface topography.

Hybridization tests of the two karyotypic forms (Form A and B) of laboratory-raised, isolines of Anopheles vagus, were conducted by induced copulation. The results of reciprocal- and back-crosses indicated that they were genetically compatible, providing viable progeny. Comparative egg morphometry and morphology, aided by scanning electron microscopy (SEM), revealed that the eggs of the two karyotypic forms were morphometrically and morphologically similar.