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.

The relationship between the abundance of Mansonia mosquitoes inhabiting a peat swamp forest and remotely sensed data.

The present study aimed to demonstrate the relationship of some environmental factors, vegetation greenness index (NDVI) and land surface temperature (LST), with the seasonal variations of Mansonia bonneae and Ma. uniformis in Khosit Subdistrict, Narathiwat Province. It was found that the Mansonia population lagged one month behind but correlated positively to NDVI, LST and rainfall. A rise in the number of mosquitoes was directly related to a rise in vegetation, temperature and rainfall.

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.

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.

Surveys for natural host plants of Mansonia mosquitoes inhabiting Toh Daeng peat swamp forest, Narathiwat Province, Thailand.

Surveys were carried out monthly from April-October 2002 to examine 68 sampling sites around "Toh Daeng" peat swamp forest in Narathiwat Province, Thailand, of which 38 were known Mansonia-positive habitats and 30 were Mansonia-negative sites. The present larval surveys were qualitative owing to features of the host plants (location, distribution, and abundance), difficulties in locating and selecting the host plants in the swamp forest, and time constraints. Twenty attempts were made for each species for larvae. The presence of Mansonia larvae on each plant species was confirmed 6 times for each plant and location. Larvae of Ma. bonneae and Ma. uniformis were obtained from 18 plant species (10 families): Metroxylon sagu, Melaleuca cajuputi, Pandanus militaris, Pandanus immerses, Hanguana malayana, Typha angustifolia, Hymenachne acutigluma, Scirpodendron ghaeri, Scleria sumatrensis, Rhynchospora corymnosa, Sacclolepis indica, Cyperus babakan, Cyperus corymbosus, Lepironia articulata, Leersia hexandra, Eichhornia crassipes, Pistia stratriotes and ferns. The emergent grasses, S. ghaeri, S. sumatrensis, H. acutigluma, R. corymnosa, S. indica, C. babakan, C. corymbosus, and L. articulata, were the preferred host plants. Samples from larger trees, M. sagu and M. cajuputi, yielded low numbers of 1-7 larvae per scraping. Ma. uniformis was recovered from most of the host plants, while Ma. bonneae preferred submerged plants and was not found on the floating aquatic plants, E. crassipes and P. stratriotes. The description of modified dipper and dipping techniques are given and discussed.

Bionomics studies of Mansonia mosquitoes inhabiting the peat swamp forest.

The present study was conducted in the years 2000-2002 to determine the bionomics of Mansonia mosquitoes, vectors of nocturnally subperiodic Brugia malayi, inhabiting the peat swamp forest, "Phru Toh Daeng", Narathiwat Province, Thailand. Fifty-four species of mosquitoes belonging to 12 genera were added, for the first time, to the list of animal fauna in the peat swamp forest. Mansonia mosquitoes were the most abundant (60-70%) by all collection methods and occurred throughout the year with a high biting density (10.5-57.8 bites per person-hour). Ma. bonneae was most prevalent (47.5%) and fed on a variety of animal hosts, including domestic cats, cows, monkeys, and man with a maximum biting density of 24.3 bites per person-hour in October. The infective bites were found for the first time in Ma. annulata collected at Ban Toh Daeng (13 00-14 00 hours) and also Ma. bonneae at forest shade (16 00-17 00 hours) and in a village (20 00-21 00 hours) with rates of 0.6, 1.1 and 1.0%, respectively. The biting activities of these two species occurred in both the day and night time, with two lower peaks at 10 00 hours (18.5 bites per person-hour) and 13 00-15 00 (8.5-10.0 bites per person-hour) hours, but the highest peak was 19 00-21 00 hours (31.5-33.0 bites per person-hour) The biting activity patterns corresponded with the periodicity found in man and domestic cats and may play an important role in either transmission or maintenance of the filarial parasites in the peat swamp forest. The relative role of Ma. bonneae and Ma. uniformis in different environmental settings (primary swamp forest and open swamp) on the transmission of nocturnally subperiodic B. malayi merits further study.

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.

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.

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).

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.

Application of GIS to the characterization of filariasis transmission in Narathiwat Province.

The geographical information system (GIS) and available survey data (both from the Filariasis Annual Reports, 1985-1999 and from the published literature) for the microfilarial infection rates are used to develop the first subdistrict-level endemicity maps of lymphatic filariasis in Narathiwat Province. The maps demonstrated the subdistrict-level geographical distribution of filariasis and the subdistricts at varied degrees of infection rate. The maps also indicate that, since 1985, there was a marked decrease in endemicity at the subdistrict level and in some areas, the infection rates were zero. However, transmission remained in the subdistricts surrounding peat swamp forest (Su-ngai Padi, Paluru, Puyo, Pasemat, Bang Khunthong, and Phron subdistricts). The house locations of infected cases, as well as the vector breeding places were geo-registered and placed as symbolic dots on the base maps obtained from Landsat's Thematic Mapper (TM) 5 and the land use map of Narathiwat to display the distribution of filariasis foci. Of 102 houses mapped, there were 40 houses in primary peat swamp forest (39.22%), 26 in rice fields (25.49%), 15 in fruit orchards (14.70%), 10 in coconut fields (9.80%) and others (10.78%). All the houses were close to the larval habitats presented in the survey. A 2-km buffer zone around the conservation boundary of primary peat swamp forest was created to locate risk areas of filariasis transmission. The buffer zone covered an area of 544.11 km2 and included 88.89% of the houses of infected cases found in 2002. It was able to identify 54 villages located in the buffer area, which might help in the determination of resource needs and resource allocation for filariasis control in Narathiwat Province.

Susceptibility and transovarial transmission of dengue virus in Aedes aegypti: a preliminary study of morphological variations.

Two types of morphological variants, the dark form and the pale form of Aedes aegypti were selected from wild-caught mosquitos. Ascertaining any differences between the two forms for susceptibility to dengue type 2 virus was performed by oral feeding. Transovarial transmission was further determined from the progenies of infected mosquitos by tracing them to the third generation. Significant differences in oral infection were not observed between these two forms of mosquitos. Transovarial transmission was found in the progenies of infected females of both forms, and the filial infection rates (FIRs) were also similar. However, there was a trend of declining FIR in the later generation. In order to achieve an accurate result, more tests are currently underway to obtain a larger number of progeny. Although the FIR was low in the present study under laboratory conditions, higher rates might occur under field conditions, which could have a significant impact on the maintenance of dengue viruses in nature.