Combined sterile insect technique and incompatible insect technique: The first proof-of-concept to suppress Aedes aegypti vector populations in semi-rural settings in Thailand.

BACKGROUND: Important arboviral diseases, such as dengue, chikungunya, and Zika virus infections, are transmitted mainly by the Aedes aegypti vector. So far, controlling this vector species with current tools and strategies has not demonstrated sustainable and significant impacts. Our main objective was to evaluate whether open field release of sterile males, produced from combining the sterile insect technique using radiation with the insect incompatible technique through Wolbachia-induced incompatibility (SIT/IIT), could suppress natural populations of Ae. aegypti in semi-rural village settings in Thailand. METHODOLOGY/PRINCIPAL FINDINGS: Irradiated Wolbachia-infected Aedes aegypti males produced by the SIT/IIT approach were completely sterile and were able to compete with the wild fertile ones. Open field release of these sterile males was conducted in an ecologically isolated village in Chachoengsao Province, eastern Thailand. House-to-house visit and media reports resulted in community acceptance and public awareness of the technology. During intervention, approximately 100-200 sterile males were released weekly in each household. After 6 months of sterile male release, a significant reduction (p<0.05) of the mean egg hatch rate (84%) and the mean number of females per household (97.30%) was achieved in the treatment areas when compared to the control ones. CONCLUSIONS/SIGNIFICANCE: Our study represents the first open field release of sterile Ae. aegypti males developed from a combined SIT/IIT approach. Entomological assessment using ovitraps, adult sticky traps, and portable vacuum aspirators confirmed the success in reducing natural populations of Ae. aegypti females in treated areas. Public awareness through media resulted in positive support for practical use of this strategy in wider areas. Further study using a systematic randomized trial is needed to determine whether this approach could have a significant impact on the diseases transmitted by Ae. aegypti vector.

Temporal and spatial distribution, sex ratio and fecundity of the eye fly siphunculina funicola (Diptera: Chloropidae) at aggregation sites during diurnal and nocturnal periods.

The present study was aimed to determine the distribution and abundance of the eye fly Siphunculina funicola (de Meijere) in Thailand and to investigate the sex ratio and fecundity of eye flies from aggregation sites collected during the day-time and night-time. The flies were collected from several provinces in central Thailand and Phuket in the south. Observations were regarding the relative abundance of eye flies in different regions and seasons. During 2007 and 2008, large populations of eye flies were noted at resting sites in central Thailand with both day and night collections. Males flies outnumbered female flies. Smaller populations were seen in Chumphon and Surat Thani Provinces with increasing numbers in Krabi and Phuket Provinces in the south. The gravid rate was nil in the few females collected in Chomphon and Surat Thani but were 3.9% and 36.3% in Krabi and Phuket, respectively. The gravid rates were higher during the dry season or during dry spells than during wet and rainy periods, suggesting egg retention by the females when oviposition sites (presumably soil) were dry. Numerous day and night collections were made in Chon Buri Province. In most collections males predominated but there was no differences in the numbers of flies collected during the two time periods. There was a slightly greater percentage of females (still lower than males) during the night collections. During the dry and hot season, due to lack of optimum oviposition sites because of dryness, the eggs were retained in the females. A series of day time collections at the end of April 2008 and in February-March 2009 had higher numbers of gravid females. Day time collections in May 2008 (start of the rainy season) showed a moderate number of gravid females, but the gravid rates were low during the rainy season, indicating higher oviposition activity by females.

The eye fly Siphunculina funicola (Diptera: Chloropidae) as a carrier of pathogenic bacteria in Thailand.

The oriental eye fly Siphunculina funicola (1.0-1.6 mm) is extremely annoying to humans and domestic animals, feeding on mucous membranes, secretions, wounds, eyes, and other moist surfaces of the host body. In many rural areas of Thailand heavy populations of this fly prevail where they aggregate on a variety of hanging substrates, such as strings, nest trailings, electrical lines, decorations, ropes, cob webs, clothes hangers, automobile radio antennae and other items in open shade close to their hosts. Both males and females feed voraciously on wounds and moist skin. With this type of persistent feeding, the eye flies are suspected to carry and transfer germs to their hosts. In the present study, bacteria were isolated from S. funicola captured from wounds, host seeking flies and from their resting sites. Some enriched and bacterial culture media were more suitable for isolation than others. A diverse group of bacteria (64 species), both gram-posi-tive and gram-negative, most in risk category 2, were identified. Bacterial colony counts from Trypic soy broth ranged from 10 to > 3.0 x 10(3) cfu/ml. The most common bacteria isolated were Acinetobacter, Aeromonas, Bacillus, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Kocuria, Pantoea, Pseudomonas, Staphylococcus and others. These bacteria may cause disease conditions in humans and animals. This is the first time bacteria from S. funicola have been reported.

Field evaluation of repellents and insecticidal aerosol compositions for repelling and control of Siphunculina funicola (Diptera: Chloropidae) on aggregation sites in Thailand.

The oriental eye fly Siphunculina funicola, a small member (1.5-1.6 mm) of the family Chloropidae, is extremely annoying to humans and domestic animals, feeding on mucous membranes, secretions, and other moist surfaces of their hosts. In central Thailand heavy populations were detected during 2006-2007 in some rural, agricultural, and periurban areas of the region. They were noted to exhibit strong synanthropy and aggregation behavior, congregating on thin (1-5 mm diam) substrates hanging in or near human habitations and animal shelters and other structures open on one or more sides. We initiated studies on the repelling and control of this eye fly at their aggregation sites, the most vulnerable targets near or in human habitations. Four formulations of repellents consisting of Everside, 3 fatty acids (with 8, 9, and 10 carbons), permethrin, and the acids C8910 plus permethrin in Everside were diluted 1:3 in tap water and sprayed on to the aggregation sites. Prior to treatment all sites including controls were disturbed to dislodge eye flies, and then the blank sites were sprayed with the aqueous suspension of the repellents. Additionally, we evaluated 3 commercially available household insecticidal aerosol formulations by treating eye flies and their resting sites (without dislodging the flies) with puffs of the aerosols. Two treatments with permethrin alone and permethrin plus acids in Nakhorn Ratchasima Province caused a complete absence of flies from the sites up to 23 h. After treatment with the repellents, the repelled flies and others were found to congregate on untreated favorable sites close to the treated ones. The repellents tested against eye flies in Chonburi Province at 2 locations caused almost complete absence of this insect on aggregation sites up to 120 h. Three household insecticide aerosols applied to aggregation sites with resting and hovering eye flies produced high to complete mortality and reduction of eye flies for 48 h or possibly longer in the treated sites. This is the first time that procedures for the evaluation of repellents and insecticides against eye flies have been tested and developed. The use of commonly available insecticide aerosol products could provide a good source of materials for the elimination of eye flies from their domestic and peridomestic aggregation sites.

Control of aggregated populations of the eye fly Siphunculina funicola (diptera: chloropidae) using pyrethroid aerosols.

Three commercially available insecticide aerosol compositions containing cyphenothrin, imiprothrin, cypermethrin, d-tetramethrin and permethrin, in various combinations and concentrations were evaluated for control of the oriental eye flies Siphunculina funicola (de Meijere) from their aggregation substrates in two villages in the Chon Buri Province, Thailand. Each aerosol produced almost complete kill of eye flies on most of the treated substrates. Time to reoccupation of each treated substrate was determined at 24, 48 and 72 hours post-treatment. All 3 aerosol preparations killed eye flies on most of the treated substrates and kept most of the substrates free of eye flies for at least 48-72 hours. The effectiveness and longevity of the aerosols varied depending on the type and location of the substrate. Smooth, hairy and polished substrates did not intercept sufficient insecticide; therefore providing low residual activity compared to porous and coated surfaces. Aerosols applied to substrates located at higher levels gave good initial kill, but reoccupation of these substrates by flies occurred relatively quickly (within 24-48 hours) following treatment. Attempts should be made to discharge aerosol flumes closer than 1 m from the aggregation substrates.

Suppression of dengue transmission by application of integrated vector control strategies at sero-positive GIS-based foci.

A serological survey of primary school children from six schools in Chachoengsao Province, Thailand, was performed at the end of the peak of dengue transmission. GIS analysis of sero-positive cases was carried out to determine transmission foci. Vector control implementation was conducted in the foci and also within 100 meters around the foci in the treated areas by community participation in collaboration with the local government. Vector control strategies included source reduction together with the use of screen covers, a combination of Bacillus thuringiensis subsp. israelensis and Mesocyclops thermocyclopoides, and lethal ovitraps. Implementation of vector control strategies in the foci was continued until the end of the rainy season. Vector control effectiveness was monitored using entomological, serological, and clinical parameters. Results showed a significant reduction of dengue vectors as well as a decrease in sero-positive children and clinical cases in treated areas when compared with untreated areas.

Pestiferous nature, resting sites, aggregation, and host-seeking behavior of the eye fly Siphunculina funicola (Diptera: Chloropidae) in Thailand.

Species of eye flies and eye gnats (Diptera: Chloropidae) are severe and persistent pests of man, domestic and wild animals, and potential vectors of pathogens. The species prevailing in the Oriental region belong to the genus Siphunculina, while those in the Neotropic and Nearctic regions belong to Liohippelates and Hippelates. These are small insects of 1-2 mm that feed on wounds, lacerations, scratches, and mucous membranes of man and higher animals. One species, Siphunculina funicola, commonly known as the Oriental eye fly, is considered the most anthropophilic in the genus, with potential involvement in the spread and mechanical transmission of infectious agents to humans and animals. Very little is known about the biology, prevalence, host-seeking, and aggregation behavior of this species in South and Southeast Asia. We initiated studies on biological aspects of this potential vector and human pest in central Thailand. The most significant findings of our study were the aggregation behaviors of S. funicola, that both sexes attack hosts, and that males outnumbered females attacking humans, dogs, and other domestic animals. They feed on wounds, scabs, lacerations, eyes, and mucous membranes. They hover around and feed on hosts during the daylight hours when host-seeking activities are more pronounced at temperatures above 25-27 degrees C under calm conditions. We noted that large masses of males and females aggregated on a variety of hanging objects such as strings, trailings, electrical lines, decorations, ropes, twines, abandoned cob webs, clothes hangers, and other hanging substrates in open shade of structures and dwellings. This behavior of eye flies brings them closer to human and animal hosts. In these aggregations, both males and females were present, with mating pairs frequently noted. In the aggregations, about 37% of the females had fully developed eggs in the rainy season, but only <1-3.6% were gravid in the hot and dry season. The average number of eggs per female was generally 42 to 44.

Community participation and appropriate technologies for dengue vector control at transmission foci in Thailand.

A community-based dengue vector control trial was conducted at transmission foci in Plaeng Yao District, Chachoengsao Province, eastern Thailand. Implementation was done by the local community in collaboration with local administration, public health, and school authorities. Our cost-effective approaches combined a source reduction campaign with appropriate vector control technologies applied within the foci (within 100 m around the foci) and also within schools attended by children from the treated areas. Vector management measures by local government included cleanup campaigns before the rainy season followed by a routine garbage pickup during the rainy season. Locally made screen covers for water jars, a combination of local Bacillus thuringiensis subsp. israelensis and Mesocyclops thermocyclopoides (copepod), and locally made lethal ovitraps were appropriate technologies used by the community in this campaign. The success of our intervention was evidenced by the significant reduction of dengue vectors and dengue hemorrhagic fever cases in treated areas compared with untreated areas.

Field evaluation of deet, Repel Care, and three plant based essential oil repellents against mosquitoes, black flies (Diptera: Simuliidae) and land leeches (Arhynchobdellida: Haemadipsidae) in Thailand.

Diethyl methyl benzamide, or deet, a commercial plant-based repellent (Repel Care), and essential ils from 3 species of plants (finger root rhizomes, guava leaves, and turmeric rhizomes), steam distillated and formulated as insect repellents, were evaluated in the field on human volunteers against hematophagous mosquitoes, black flies, and land leeches in Thailand. Field trials were conducted against wild mosquitoes in Bang Bua Thong District, Nonthaburi Province, and in the Thap Lan National Park Headquarters, Nadee District, Pranchinburi Province; anthroophilic black flies (Diptera: Simuliidae) at the Forestry Fire Control Station in Doi Inthanon National Park, Chomthong district, Chiang Mai Province; and land leeches (Arhynchobdellida: Haemadipsidae) in the Khao Yai National Park, Pak Chong District, Nakhon Ratchasima Province. The 3 experimental plant-based essential oil formulations as well as Repel Care and deet provided complete protection from mosquito landing and biting for up to 9 h (duration of the experiment). Similar results were obtained with the 5 products against black flies, providing 100% protection for 9 h but 96-82% protection after 10 and 11 h posttreatment. The 5 repellent products also provided 100% protection against land leeches for at least 8 h. Thi is the 1st report of repellency of plant-based repellents against black flies and land leeches in Thailand. The identification and availability of inexpensive sources of plant-based oils, i.e., finger root rhizomes, guava leaves, and turmeric rhizomes providing long-lasting repellency against blood-sucking organisms are promising leads into commercial production of relatively safe and effective repellents.

Mosquito larvicidal activity of aqueous extracts of long pepper (Piper retrofractum vahl) from Thailand.

Aqueous extracts of nine medicinal plants were bioassayed against larvae of Culex quinquefasciatus Say and Aedes aegypt (L.). Among these plants, the long pepper, Piper retrofractum Vahl (Piperaceae), showed the highest level of activity against mosquito larvae. To gain more information on larvicidal activity of P. retrofractum, fresh fruits of this plant were extracted in water and the extracts made into powder and bioassayed against 3rd and 4th instar larvae of Cx. quinquefasciatus and Ae. aegypti in the laboratory. Extracts of unripe (001/3) and ripe (002/3 and 001/4) fruits showed different levels of activity against Cx. quinquefasciatus larvae. Extracts 001/3 and 002/3 were equi-toxic to a Bacillus sphaericus resistant and susceptible strains, both from Thailand. The ripe fruit extract 002/3 was somewhat more active against Ae. aegypti than Cx. quinquefasciatus. Another ripe fruit extract (001/4) was much more toxic to both mosquito species. Diluted solutions of the solid extract (002/3) in distilled water lost their larvicidal activity upon aging. Loss of activity at 25 degrees C was greater than that stored at 4 degrees C, and greater in water than in acetone solution.

Combination of Mesocyclops thermocyclopoides and Bacillus thuringiensis var. israelensis: a better approach for the control of Aedes aegypti larvae in water containers.

The efficacy of a local Thai-strain of the copepod, Mesocyclops thermocyclopoides and the larvicide, Bacillus thuringiensis var. israelensis (Bti), used jointly and singly, was studied against Aedes aegypti in water containers. In a laboratory test, copepods alone produced mortality of 98-100% in 1st instar larvae of Ae. aegypti at copepod:larvae ratios ranging from 1:1 to 1:4. In an outdoor field simulated experiment that ran for 16 wk, after a single inoculation, the treatment of copepods and Bti combined yielded the better, more sustainable results than the agents used individually. Numbers of mosquito larvae per sample in the combined treatment were zero during the first 8 wk; larval numbers then increased but were maintained at a very low level for the next 4 wk after which the larval numbers increased moderately but still remained below numbers in the control. Bti alone kept the larvae at the zero level for the first 4 wk after which their numbers increased slightly and were at low levels up to 12 wk. Copepods alone maintained larval numbers at a low level as compared with those of the control. During the course of the experiment the larval numbers in the control were greater than 20 per sample. Statistically significant differences were noted among treatment means (F = 23.083, df = 3/60, P<0.01) over the total period of the study. The number of copepods in the joint treatment was significantly higher than in the copepod alone treatment for the first 8 wk (t = -4.97, df = 14, P<0.01). The density of copepods, however, for the whole 16-wk period was not significantly different in these two treatments (t = -1.51, df = 30, P>0.1).