Susceptibility of Aedes flavopictus miyarai and Aedes galloisi mosquito species in Japan to dengue type 2 virus

Objective: To evaluate the potential of local mosquitoes to act as vectors for dengue transmission in Japan. Methods: Serotype 2 ThNH28/93 was used to test the dengue susceptibility profiles of Aedes flavopictus miyarai (Ae. f. miyarai), Aedes galloisi (Ae. galloisi) and Aedes albopictus (Ae. albopictus), which were collected in Japan. We used Aedes aegypti from Thailand as a positive control. The mosquitoes were infected with the virus intrathoracically or orally. At 10 or 14 days post infection, the mosquitoes were dissected and total RNA was extracted from their abdomens, thoraxes, heads and legs. Mosquito susceptibility to dengue virus was evaluated using RT-PCR with dengue virus-specific primers. Differences in the infection and mortality rates of the different mosquito species were tested using Fisher's exact probability test. Results: The infection rates for dengue virus administered intrathoracically to Ae. f. miyarai, Ae. galloisi and Aedes aegypti mosquitoes were identical by RT-PCR on Day 10 post infection. All of the body parts we tested were RT-PCR-positive for dengue virus. For the orally admin-istered virus, the infection rates in the different body parts of the Ae. f. miyarai mosquitoes were slightly higher than those of Ae. albopictus mosquitoes, but were similar to the control mosquitoes (P>0.05). The mortality rates for Ae. f. miyarai and Ae. albopictus mosquitoes were similar (P=0.19). Our data indicated that dengue virus was able to replicate and disseminate to secondary infection sites in all of the four mosquito species (Japanese and Thai). Conclusions: Ae. albopictus is a well-known candidate for dengue transmission in Japan. However, our data suggest that Ae. f. miyarai from Ishigaki Island (near Okinawa Island) and Ae. galloisi from Hokkaido (Northern Japan) should also be regarded as potential vectors for dengue transmission in these regions. Further studies on these mosquitoes should be conducted.

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.

Residual effects of Mossmann 100 (permethrin 10% EC) impregnated bed nets and its impact on malaria vectors and incidence of malaria.

The objective of this field trial was to assess the residual effectiveness of permethrin 10% treated mosquito nets on malaria control compared with untreated nets. The study was carried out between July and December 2007 in the Pong Nam Ron District of Chantaburi Province, Thailand. Mosquito population densities were assessed using the landing catch method. Mosquitoes were collected between 6:00 PM and 12:00 PM. Residual effectiveness of the treated nets was assessed using standard WHO bioassay tests carried out monthly using Anopheles dirus mosquitoes reared in the insectary of the Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Thailand. The results showed the population densities of Anopheles spp, including the malaria vector Anopheles minimus, were unaffected in the study area where mosquito nets treated with Mossmann 100 (permethrin 10% EC) at 300 mg/m2 were used. WHO bioassay tests showed the nets treated with Mossmann 100 remained biologically effective against An. dirus for up to six months. Indigenous cases of malaria were reduced by 27.7% at the site where the nets treated with Mossman 100 (permethrin 10%EC) were used but no changes in malaria cases at the control site were seen.

Repellent activity of essential oils against cockroaches (Dictyoptera: Blattidae, Blattellidae, and Blaberidae) in Thailand.

Seven commercial essential oils extracted from the plant species Boesenbergia rotunda (L.) Mansf., Citrus hystrix DC., Curcuma longa L., Litsea cubeba (Lour.) Pers., Piper nigrum L., Psidium guajava L. and Zingiber officinale Roscoe, and naphthalene as a control, were evaluated for repellent activity against the three cockroach species Periplaneta americana (L.), Blattella germanica (L.) and Neostylopyga rhombifolia (Stoll) under laboratory conditions. The essential oil derived from Citrus hystrix showed the best repellency over other candidate essential oils and naphthalene. The essential oil of Citrus hystrix exhibited complete repellency (100%) against P. americana and B. germanica, and also showed the highest repellency (among the essential oils tested) of about 87.5% against N. rhombifolia under laboratory conditions. In the field, Citrus hystrix essential oil formulated as a 20% active ingredient in ethanol and some additives provided satisfactory repellency of up to 86% reduction in cockroaches, mostly P. americana and N. rhombifolia with a residual effect lasting a week after treatment. Citrus hystrix essential oil has good potential for being used as a cockroach repellent. Further improvements in efficacy and residual activity may be realized with appropriate formulations.

Repellency of essential oils extracted from plants in Thailand against four mosquito vectors (Diptera: Culicidae) and oviposition deterrent effects against Aedes aegypti (Diptera: Culicidae).

In this study we evaluated and reported repellent effects of essential oils from Thai plants against 4 mosquito vectors: Aedes aegypti, Ae. albopictus, Anopheles. dirus and Culex quinquefasciatus under laboratory conditions using human volunteers. The essential oils were extracted from 18 plant species, belonging to 11 families, and the oils were then prepared as 10% solution in absolute ethanol with additives. Two chemical repellents, deet and IR3535, were also prepared in the same formulation as the essential oil repellents and tested for repellency as controls. The essential oils were also evaluated for oviposition deterrent effects against Ae. aegypti under laboratory conditions. The results show night-biting mosquitoes (An. dirus and Cx. quinquefasciatus) and Ae. albopictus were more sensitive to all the essential oils (repellency 4.5 - 8 hours) than was Ae. aegypti (repellency 0.3 - 2.8 hours), whereas deet and IR3535 provided excellent repellency against all four mosquito species (repellency 6.7- 8 hours). All essential oils exhibited oviposition deterrent activity against Ae. aegypti with various degrees of repellency ranging from 16.6 to 94.7%, whereas deet and IR3535 had no repellency. The present study demonstrates the potential for using essential oils as mosquito repellents and oviposition deterrents. These findings may lead to new and more effective strategies for protection from and control of 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.

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.

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.

Susceptibility of two karyotypic forms of Anopheles aconitus (Diptera: Culicidae) to Plasmodium falciparum and P. vivax

Quatro colônias desenvolvidas em laboratório, de duas formas cariotípicas de Anopheles aconitus i.e. forma B (cepa Chiang Mai e Phet Buri) e C (Cepa Chiang Mai e Mae Hong Son), foram infectadas experimentalmente com Plasmodium falciparum e P. vivax usando técnica de alimentação com membrana artificial e dissecados oito e 12 dias após alimentação da média de oocistos e esporozoitos, respectivamente. Os resultados revelaram que An. aconitus formas B e C foram suscetíveis ao P. falciparum e P. vivax isto é, forma B (cepa Chiang Mai e Phet Buri/P. falciparum e P. vivax) e forma C (cepa Chiang Mai e Mae Hong Son/P. vivax). Análises estatísticas comparativas das taxas de oocistos, número médio de oocistos por intestino médio infectado e taxas de esporozoitos entre todas as cepas de An. aconitus formas B e C ao grupo interno de vetores controles, An. minimus A e C, não exibiram nenhuma diferença significante, confirmando o alto potencial vetor das duas espécies de Plamodium. Os cristais semelhantes a esporozoitos encontrados no lobo médio das glândulas salivares que poderiam ser um fator enganoso na identificação de esporozoitos verdadeiros nas glândulas salivares foram encontrados em ambos An. aconitus formas B e C.

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 colonization of Aedes lineatopennis.

Aedes lineatopennis, a species member of the subgenus Neomelaniconion, could be colonized for more than 10 successive generations from 30 egg batches [totally 2,075 (34-98) eggs] of wild-caught females. The oviposited eggs needed to be incubated in a moisture chamber for at least 7 days to complete embryonation and, following immersion in 0.25-2% hay-fermented water, 61-66% of them hatched after hatching stimulation. Larvae were easily reared in 0.25-1% hay-fermented water, with suspended powder of equal weight of wheat germ, dry yeast, and oatmeal provided as food. Larval development was complete after 4-6 days. The pupal stage lasted 3-4 days when nearly all pupae reached the adult stage (87-91%). The adults had to mate artificially, and 5-day-old males proved to be the best age for induced copulation. Three to five-day-old females, which were kept in a paper cup, were fed easily on blood from an anesthetized golden hamster that was placed on the top-screen. The average number of eggs per gravid female was 63.56 +/- 22.93 (22-110). Unfed females and males, which were kept in a paper cup and fed on 5% multivitamin syrup solution, lived up to 43.17 +/- 12.63 (9-69) and 15.90 +/- 7.24 (2-39) days, respectively, in insectarium conditions of 27 +/- 2 degrees C and 70-80% relative humidity.

Sequence analyses of three nuclear ribosomal loci and a mitochondrial locus in cytologically different forms of Thai Anopheles aconitus mosquitoes.

Ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of Anopheles aconitus mosquitoes were examined to investigate intra- and inter-species variation amongst the members of the Minimus group of Anopheles subgenus Cellia. Three rDNA loci (ITS1, ITS2 and D3 regions) and a mtDNA locus (cytochrome oxidase II) were analyzed in An. aconitus Form B and Form C collected in Chiang Mai Province, Thailand. The results show that the consensus sequences of the four loci of the two forms are consistent with those of mosquitoes in the genus Anopheles. No intraindividual variation was detected, but intrapopulation variation was present with polymorphic sequences in some forms for each gene examined. The variation rates were approximately 0.15 to 0.8%. These data indicate that An. aconitus Form B and Form C in Chiang Mai, Thailand are conspecific. In this study, the complete ITS1 sequence of An. aconitus is reported for the first time. The region showed a high variation rate (approximately 55%), compared to the closely related species An. minimus C. It is suggested that this rDNA locus may provide sequence information to differentiate the members of the Minimus group of Anopheles subgenus Cellia.

Comparative morphometry and morphology of Anopheles aconitus Form B and C eggs under scanning electron microscope

Estudos comparativos morfométricos e morfológicos de ovos à microscopia eletrônica de varredura (SEM) foram efetuados nas três linhagens de duas formas cariotípicas de Anopheles aconitus, isto é, Forma B (linhagens Chiang Mai e Phet Buri) e Forma C (linhagens Chiang Mai e Mae Hong Son). Exame morfométrico revelou a variação intraespecífica com respeito à largura de superfície [36,77 ± 2,30 µm (Forma C: linhagem Chiang Mai) = 38,49 ± 2,78 µm (Forma B: linhagem Chiang Mai) = 39,06 ± 2,37 µm (Forma B: linhagem Phet Buri) > 32,40 ± 3,52 µm (Forma C: linhagem Mae Hong Son)] e número de tubérculos posteriores sobre a superfície livre [2,40 ± 0,52 (Forma B: linhagem Phet Buri) = 2,70 ± 0,82 (Forma B: linhagem Chiang Mai) < 3,10 ± 0,32 (Forma C: linhagem Chiang Mai) = 3,20 ± 0,42 (Forma C: linhagem Mae Hong Son)] embora a topografia de superfície dos ovos entre as três linhagens de duas formas cariotípicas tenham sido morfologicamente semelhantes.

Zymogram patterns of Naegleria spp isolated from natural water sources in Taling Chan district, Bangkok.

A genetic approach was cited for species detection of the ameba genus Naegleria using allozyme electrophoresis to characterize the trophozoite stage of three strains of Naegleria fowleri isolated from patients with primary amebic meningoencephalitis, five thermophilic (45 degrees C) Naegleria spp isolated from natural water sources in the Taling Chan district, and a reference control strain, Naegleria fowleri CDC VO 3081. Isoenzymes of ameba whole-cell extracts were analyzed by vertical polyacrylamide slab gel electrophoresis to determine whether there was any correlation between different strains of the ameba. The results showed that five out of fifteen enzymes; aldehyde oxidase (ALDOX), aldolase (ALD), a-glycerophosphate dehydrogenase (a-GPDH), xanthine dehydrogenase (XDH), and glutamate oxaloacetate transaminase (GOT), were undetectable in the pathogenic strains, while the other enzymes; esterase (EST), fumerase (FUM), glucose-6-phosphate dehydrogenase (G-6-PDH), glucose phosphate isomerase (GPI), isocitate dehydrogenase (IDH), lactate dehydrogenase (LDH), leucine aminopeptidase (LAP), malic enzyme (ME), glucose phosphomutase (GPM), and malate dehydrogenase (MDH), were detected. Naegleria fowleri strains were biochemically the most homogeneous. They showed intraspecific isoenzyme variation that allowed them to be grouped. In contrast, the allozyme patterns (EST 1-7, IDH) of Naegleria spp isolated from the environment showed interspecific isoenzyme variations from the pathogenic Naegleria strain. In conclusion, this study recognized the zymograms of the Naegleria fowleri strains were heterogenically different from the thermophilic 45 degrees C Naegleria spp isolated from the environment.

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.

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.