Comparison between Musca domestica and Chrysomya megacephala as carriers of bacteria in northern Thailand.

A comparison between the common house fly, Musca domestica, and the Oriental latrine fly, Chrysomya megacephala, was assessed for their potential as carriers of bacteria in urban areas of Chiang Mai Province, northern Thailand. C. megacephala was significantly more likely to carry bacterial species than M. domestica; however, no significant difference was found between the number of positive male and female flies within the same species. A total of 42 bacterial species were isolated. The most common bacterium isolated from M. domestica was coagulase-negative staphylococci (n=57) followed by Escherichia coli (n=10) and Viridans streptococci (n=10), while that of C. megacephala was non-fermentative gram-negative bacilli (n=59) followed by coagulase-negative staphylococci (n=54).

Bioactivity of citrus seed for mosquito-borne diseases larval control.

This study aimed to determine the activity of citrus-seed extract against Ae. aegypti and Cx. quinquefasciatus larvae. The results indicated that ethanol citrus-seed extract showed the best killing effect on Ae. aegypti larvae, followed by local liquor, and water, with LC50 of 2,267.71, 6,389.22, and 135,319.40 ppm, respectively, whereas against Cx. quinquefasciatus larvae, the LC50 were 2,639.27, 5,611.66, and 127,411.88 ppm, respectively. Temephos was tested against Ae. aegypti larvae; the LC50 was 0.00057 ppm, which was nearly 4,000,000 times less than ethanol citrus-seed extract. When ethanol citrus-seed extract and temephos were tested with Nile tilapia (Oreochromis niloticus), a standard environmental organism, using LC50 of Ae. aegypti larvae at 2,267 and 0.00057 ppm, respectively, fish mortality was 0%. The results suggested that ethanol citrus-seed extract had no harmful effect on the fish, and that temephos, which is recommended by WHO, was safe for use in drinking water. However, when the LC50 dose that killed Ae. aegypti larvae for local liquor (6,389 ppm) and water extract (135,319 ppm) were tested with fish, the mortality rates were 35% and 100%, respectively. On the whole, the results suggested that ethanol citrus-seed extract is environmentally friendly and can be used in the control of Ae. aegypti and Cx. quinquefasciatus larvae.

Formulation of tablets from the crude extract of Rhinacanthus nasutus (Thai local plant) against Aedes aegypti and Culex quinquefasciatus larvae: a preliminary study.

Dried root powder of Rhinacanthus nasutus, Thong Phan Chang (Thai name) were extracted with methanol (MeOH) in a Soxhlet apparatus and made into 2 formulations of tablet containing the extract at 5% and 10% concentration. Due to the viscous and poor flow properties of the crude MeOH extract obtained, a wet granulation method was conducted in developing the tablets. Lactose was used as a filler. Polyvinyl pyrrolidone (PVP) K30 (15% w/w solution in alcohol) was used as the binding agent, while stearic acid (2% w/w) was used as a lubricant. Both formulas of prepared tablets had a smooth shiny surface with a round shape. Other physical properties of the tablets, such as weight variation, friability and disintegration time, met the requirements of the USP XX standard. The mosquito larvicidal activity of prepared tablets containing 5% and 10% R. nasutus extract against Aedes aegypti were not significantly different from each other (p > 0.05), with 48-hour LC50 values of 13.6 and 14.2 mg/I for the 5% and 10% tablets, respectively, while their activities against Culex quinquefasciatus were similar (p > 0.05) with LC50 values of 18.7 and 17.3, respectively. The larvicidal activity levels against Ae. aegypti and Cx. quinquefasciatus were also not significantly different from each other (p > 0.05). No larval mortality was observed in the two control groups: lactose solution and dechlorinated water. Toxicity to female and male fish (Poecilia reticulata) was tested with the prepared tablets. The toxicity of tablets containing 5% and 10% extracts were not significantly different from each other for the P. reticulata females with 48-hour LC50 values of 105.2 and 110.8 mg/I, respectively, and for P. reticulata males with LC50 values of 99.1 and 103.4 mg/I, respectively. Female and male P. reticulata were sensitive to the same dose of the extract. No fish died in the two control groups, with lactose solution and dechlorinated water. Acute-toxicity bioassay with fish showed that with an exposure of 48 hours the LC50 values of the tablets containing 5% and 10% were 5- to 10-fold higher than the LC50 of R. nasutus against mosquito larvae. These prepared tablets could possibly used to control mosquito vectors and be introduced into the mosquito control program.

Efficacy of repellent products against caged and free flying Anopheles stephensi mosquitoes.

The efficacy of 9 repellents (8 commercial repellents and one product under development) was evaluated on the skin at dosages of 0.65 and 1.7 mg of product/cm2, the latter dosage being the industrial standard for deet based repellents. The repellents were applied to the arm or lower leg of a human subject and tested against Anopheles stephensi in a cage or flying freely in a mosquito-proof room. In the cage tests, a product with 20% p-menthane-3, 8-diol (PMD) active ingredient provided complete repellency for 7-8 hours, while with 10% PMD had complete repellency for only 30 minutes. The natural oils of clove (Syzygium aromaticum) (10% active ingredient) plus makaen (Zanthoxylum limonella) (10% active ingredient) gave protection for 4-5 hours. In the case of free flying mosquitoes, products with 20% and 30% PMD gave complete protection for 11-12 hours at a dosage of 1.7 mg/cm2 or 6 hours at half the dosage, while the product with 10% PMD afforded protection for less than 2 hours. At the higher dosage rate 40% citronella and hydroxyethyl isobutyl piperidine carboxylate, a new synthetic compound, provided complete repellency for 7 hours. Fifty percent deet (N,N-diethyl-3-methylbenzamide) was effective for 30 hours if left undisturbed on the skin.

Screening for larvicidal activity in some Thai plants against four mosquito vector species.

Ninety-six ethanolic extracts from various parts of 84 Thai plant species were tested for their larvicidal activity against Aedes aegypti mosquitoes. Extracts from Rhinacanthus nasutus, Derris elliptica, Trigonostemon reidioides, Homalomena aromatica, Stemona tuberosa and Acorus calamus possessed high larvicidal activity, with LC50 values between 16.0 and 48.2 mg/l. Petroleum ether (PE) and methanol (MeOH) extracts were tested for their larvicidal activity against 4 mosquito vector species. The PE extract of R. nasutus exhibited larvicidal effects against Ae. aegypti, Culex quinquefasciatus, Anopheles dirus and Mansonia uniformis with LC50 values between 3.9 and 11.5 mg/l, while the MeOH extract gave LC50 values of between 8.1 and 14.7 mg/l. D. elliptica PE extract showed LC50 values of between 11.2 and 18.84 mg/l and the MeOH extract exhibited LC50 values between 13.2 and 45.2 mg/l.

Laboratory and field trial of developing medicinal local Thai plant products against four species of mosquito vectors.

Oils of Syzygium aromaticum (clove) and Zanthoxylum limonella (makaen), widely used essential oils for dental caries or flavoring of food in Thailand, were prepared as 10 experimental repellent products in gel or cream form against Aedes aegypti, Culex quinquefasciatus, and Anopheles dirus under laboratory conditions, using the human-arm-in-cage method. Two products that gave the longest-lasting complete protection were selected to examine their repellency against a variety of mosquito species under field conditions. In laboratory tests, 0.1 g of each product was applied to 3x10 cm of exposed area on a volunteer's forearm, while in field trials, 1.0 g was applied to each volunteer's leg (from knee to ankle). In the laboratory, the gel dosage form contained 20% clove oil (Gel B) or 10% clove plus 10% makaen oil mixture (Gel E) were promising plant-based repellents against three mosquito species and gave significantly longer complete protection times of 4-5 hours than all other developing products. Therefore, their efficacy in the field was evaluated. Under field conditions, Gel E showed complete protection for 4 hours and gave 95.7% repellency after 5 hours application, whereas Gel B and 20% deet (di-methyl benzamide) provided only 86.8 and 82.7% repellency after treatment, respectively against Ae. aegypti, daytime-biting mosquitos. For nighttime-biting, the 3 repellents under development yielded equally excellent (average 97.1%) repellency for 5 hours against the predominant Cx. quinquefasciatus and Mansonia uniformis, but they gave 89.0% repellency against Cx. tritaeniorhynchus and Cx. gelidus. This finding demonstrated the effectiveness of Gel B and Gel E products for possible use by low-income rural communities against various mosquito species.

Temephos resistance in two forms of Aedes aegypti and its significance for the resistance mechanism.

Aedes aegypti, at the larval stage, has been subjected to the temephos selection in laboratory. The level of temephos resistance was detected in a microplate by biochemical assay using WHO bioassay technique. The major enzyme-based resistance mechanisms involved in temephos resistance include elevated nonspecific esterase, oxidase and insensitive acetylcholinesterase. After 19 generations of temephos selection, the selected group showed resistance ratios of 4.64 and 16.92, when compared with a non-selected group and the WHO susceptible strain, respectively. The two seperated forms, type form and the pale form of Ae. aegypti showed low levels of resistance to temephos after 19 generations of selection, with resistance ratios of 4.82 and 4.07 for the type form and the pale form, respectively; when compared with the non-selected strain, 17.58 and 14.84, when compared with the WHO susceptible strain. This showed that the type form could develop higher level resistance than the pale form. The esterase inhibitor (S,S,S-tributyl phosphorotrithioate, DEF) or synergist implicated detoxifying esterase in all the temephos selected groups and the presence of elevated esterase were confirmed by biochemical assay. There were significant differences in elevated esterase activity between the temephos selected groups and the non-selected group. However no significant difference between the type form and the pale form was found. Besides the elevated esterase, there was no change in monooxygenase activity and no evidence of insensitive acetylcholinesterease for all temephos selected groups. These results suggest that temephos resistance could be developed in Ae. aegypti under selection pressure and that the main mechanism is based only on esterase detoxification.

Seroepidemiological survey among schoolchildren during the 2000-2001 dengue outbreak of Ratchaburi Province, Thailand.

From August 2000 to 2001, a dengue outbreak occurred in Mueang district, Ratchaburi Province, Thailand. About 800 cases of dengue infection were reported, and among them, 49.5% were clinically diagnosed as dengue hemorrhagic fever according to the WHO criteria. During the outbreak, the incidence rate of dengue infection in Hin Gong subdistrict was 2.9 per 1,000 population. A seroepidemiological survey was conducted among primary schoolchildren from July 2000 to June 2001, to monitor dengue transmission. In a baseline survey, 283 children were surveyed for dengue antibody and 71% were IgG seropositive. In June 2001, the rate of dengue infection showed an increase of 8.8% with 8.0% among immune children and 10.3% among naive schoolchildren. Among 283 schoolchildren, 90 were followed up 3 times, in September and December 2000, and June 2001. An increase in the rate of seroconversion was observed in the period September to December 2000, while the peak dengue outbreaks in the dry season occurred in February 2001. Serosurveys among schoolchildren appear to be early warning system, and can be advantageous in early dengue control actions, in order to break the chain of transmission before an impending epidemic.

Potential development of temephos resistance in Aedes aegypti related to its mechanism and susceptibility to dengue virus.

The addition of temephos to water containers as a larvicide against Aedes aegypti was commonly used as a part of DHF control programs. The widespread, or long-term, application of insecticides can lead to the development of mosquito resistance to the insecticides through selection pressure. This presents a problem for disease control. Therefore, this study was conducted in the laboratory to observe the potential development of resistance to temephos and the mechanism involved in Ae. aegypti, and to study the significance for dengue infection. The larvae were selected in consecutive generations. The level of resistance to temephos was detected by WHO assay technique. After 19 generations of selection, a low level of resistance was found. The resistance ratio at LC50 was 4.64 when compared with the non-selected group. The assay for major enzyme-based resistance mechanisms was done in a microtiter plate to detect elevated non-specific esterases, monooxygenase, and insensitive acetylcholinesterase in the temephos-selected and non-selected groups. It revealed a significant increase in esterase activity when compared with the non-selected group. There was no elevation of monooxygenase or insensitive acetylcholinesterase activities. However, when an esterase inhibitor (S, S, S-tributyl phosphorotrithioate, or DEF) was added to temephos and the susceptibility in the selected group was studied, the resistance ratio was reduced from 16.92 to 3.57 when compared with a standard susceptible strain (Bora Bora). This indicates that the esterases play an important role in temephos resistance. Dengue-2 virus susceptibility was studied by oral feeding to females of the temephos-selected (S19) and the non-selected groups. The dissemination rates, when the titer of virus in the blood meal was 7.30 MID50/ml, were 11.11% and 9.38% for the selected and non-selected groups, respectively. When the titer of virus in the blood meal was 8.15 MID50/ml, the dissemination rates increased to 24.24% and 33.33%, respectively. A statistical difference in viral susceptibility was not found between the two groups. This suggested that the low level of temephos resistance might not affect oral susceptibility. However, this needs further study.

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.

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.