Search

We evaluated the behavioral responses of Aedes albopictus and Anopheles minimus to 3 isovaleric acid and lactic acid-based chemical lure blends and 2 individual alcohols, using Spatial Repellency Assay in a high-throughput screening system (HITSS). Five doses of 0.0002, 0.001, 0.0025, 0.005, and 0.01 g were tested per lure. A BG-lure was used as a reference standard. After 10-min exposure, the number of mosquitoes moving toward or away from the treated chamber was calculated. The results showed that all lures were repellent against Ae. albopictus except for Lure-4 (4% w/v isovaleric acid + 2% w/v lactic acid + 0.0025% w/v myristic acid + 2.5% w/v ammonium hydroxide) which showed a nonsignificant attractancy at the lowest dose. Significantly high spatial repellency was observed at the highest dose of all the tested lures including BG-lure. Lure-2 (isoamyl alcohol) was significantly repellent at all the tested doses. Against An. minimus, Lure-5 (0.02% w/v isovaleric acid + 2% w/v lactic acid) showed significant spatial repellency while Lure-4 was significantly attractant, at all the tested doses. All lures, except Lure-4, showed strong spatial repellency at high doses and attractancy or weak spatial repellency at the lowest dose of 0.0002 g. In summary, our study demonstrated that spatial repellency and attractancy of the tested lures were influenced by both the dose tested and the mosquito species. Lure-2 and Lure-4 are potential spatial repellents and attractants, respectively, for malaria and dengue vectors. However, further studies are necessary to confirm these results at a semifield and open field level.

Contact irritant (locomotor excitation) and noncontact spatial repellent avoidance behavior to deltamethrin and cypermethrin at dosages 0.025, 0.05, and 0.1 g/m2 impregnated on papers were evaluated in the laboratory against deltamethrin- and cypermethrin-resistant field populations of female Aedes aegypti (L.) from Rayong and Chanthaburi Provinces, Thailand. Pyrethroid-resistant populations were compared with a susceptible laboratory strain (NIH-Thai) using an ‘excito-repellency’ (ER) test system. Both NIH-Thai and field mosquitoes had stronger contact irritancy responses compared to the relatively weak noncontact repellency effects. Contact assays with deltamethrin and cypermethrin at 0.1 g/m2 showed high escape rates for Rayong (80.1 and 83.4%, respectively) and Chanthaburi (84.6 and 73.1%, respectively) mosquitoes. Cypermethrin produced significantly different (P < 0.05) percent escape responses in contact tests between NIH-Thai and field mosquitoes. Only deltamethrin contact at 0.05 g/m2 produced a significant escape response (P < 0.001) between NIH-Thai and Rayong mosquitoes. These results suggest that there may not be an overall significant effect of background pyrethroid resistance on escape response, and the differences by comparisons may reflect inherent individual variation when using the ER bioassay system. The results show that pyrethroid resistance in Ae. aegypti does not appear to influence or reduce contact avoidance responses with the compounds tested. In particular, deltamethrin at 0.1 g/m2 was an effective contact irritant and toxic compound against pyrethroid-resistant populations of Ae. aegypti. Therefore, 0.1 g/m2 deltamethrin could be considered for residual applications of either fixed surfaces or materials (e.g., curtains) as a supplemental control measure against adult dengue vectors.

Species of the Anopheles barbirostris complex (Myzorhynchus Series of the subgenus Anopheles) are potential vectors of malaria and filariasis parasites. Owing to the lack of reliable identification methods, the biting activity and host preference of the species within this complex have not been previous described. In this study, the trophic behavior and host preferences of the species in the complex were determined in Thailand, and a map of their geographical distributions constructed. Adult female mosquitoes in Chanthaburi and Prachuap Khiri Khan provinces were collected using human landings (indoor and outdoor) and cow-bait. Morphological characters were used to initially identify the mosquitoes, and then, species were further separated by multiplex PCR. Four species were identified in the An. barbirostris complex: An. wejchoochotei Taai & Harbach (Diptera: Culicidae), An. saeungae Taai & Harbach (Diptera: Culicidae), An. dissidens Taai & Harbach (Diptera: Culicidae), and An. campestris Reid (Diptera: Culicidae). The species showed exophagic and zoophilic behavior, except An. wejchoochotei in Chanthaburi, which displayed slight endophilic behavior. Indoor and outdoor blood feeding behavior of An. wejchoochotei and An. saeungae peaked at 2000 h. An updated distribution map of the An. barbirostris complex in Thailand is presented. With a better understanding of the sibling vector species and their spatial distributions, more specific and effective control strategies for vectors of malaria and filariasis in Thailand can be achieved.

Aedes aegypti is the primary vector of dengue viruses in Thailand. Control of this mosquito continues to rely heavily on use of insecticides in various forms and applications. The synergistic effect of piperonyl butoxide (PBO), combined with deltamethrin against eight populations of Ae. aegypti collected from different regions in Thailand is presented. The standard WHO adult contact bioassays found all populations with low to moderate levels of resistance to deltamethrin alone (using a 0.05% discriminating concentration), with final mortalities ranging from 15.6 to 70%, while a laboratory strain was fully susceptible (100% mortality). Pre-exposure of female mosquitoes to 4% PBO for 1 h, followed immediately by exposure to deltamethrin for 1 h, significantly increased mortality in seven populations (64.8–98.1%) with the exception of mosquitoes derived from Lampang Province. The knockdown time (KDT) synergist ratios between deltamethrin only and PBO + deltamethrin ranged from 1.7 to 2.8 for KDT50 and 1.9 to 4.0 for KDT95. Between deltamethrin alone and mosquitoes exposed to PBO + deltamethrin, all resistant populations produced significant differences (P

An unprecedented malaria outbreak occurred in Ubon Ratchathani Province, northeastern Thailand, in 2014. The province showed the highest number of malaria cases of all Thai provinces. Five entomological surveys were conducted at 8 sentinel sites from September 2013 to September 2015 to address the role of different Anopheles species in malaria transmission. Mosquito collections were conducted using human landing catches and cow bait. A total of 10,369 Anopheles mosquitoes were collected and 2,240 were morphologically identified as potential malaria vectors, including An. dirus (n = 78), An. minimus (n = 18), An. sawadwongporni (n = 4), An. barbirostris s.l. (n = 819), An. philippinensis (n = 612), An. nivipes (n = 676), An. annularis (n = 42), An. aconitus (n = 7), and An. rampae (n = 142). Real-time polymerase chain reaction was used to screen for the presence of Plasmodium spp. in salivary glands. The proportion of primary vectors of surveyed villages was very low (

Additional file 1: Table S1. Collection locations of the Hyrcanus Group associated environment in Ubon Ratchathani Province, Thailand.

Background Members of the Anopheles hyrcanus group have been incriminated as important malaria vectors. This study aims to identify the species and explore the insecticide susceptibility profile within the Anopheles hyrcanus group in Ubon Ratchathani Province, northeastern Thailand where increasing numbers of malaria cases were reported in 2014. Methods Between 2013 and 2015, five rounds of mosquito collections were conducted using human landing and cattle bait techniques during both the rainy and dry seasons. Anopheles mosquitoes were morphologically identified and their insecticide susceptibility status was investigated. Synergist bioassays were carried out with An. hyrcanus (s.l.) due to their resistance to all insecticides. An ITS2-PCR assay was conducted to identify to species the Hyrcanus group specimens. Results Out of 10,361 Anopheles females collected, representing 18 taxa in 2 subgenera, 71.8% were morphologically identified as belonging to the Hyrcanus Group (subgenus Anopheles), followed by An. barbirostris group (7.9%), An. nivipes (6.5%), An. philippinensis (5.9%) and the other 14 Anopheles species. Specimens of the Hyrcanus Group were more prevalent during the rainy season and were found to be highly zoophilic. Anopheles hyrcanus (s.l.) was active throughout the night, with an early peak of activity between 18:00 h and 21:00 h. ITS2-PCR assay conducted on 603 DNA samples from specimens within the Hyrcanus Group showed the presence of five sisters species. Anopheles peditaeniatus was the most abundant species (90.5%, n = 546), followed by An. nitidus (4.5%, n = 27), An. nigerrimus (4.3%, n = 26), An. argyropus (0.5%, n = 3), and An. sinensis (0.2%, n = 1). All An. hyrcanus (s.l.) specimens that were found resistant to insecticides (deltamethrin 0.05%, permethrin 0.75% and DDT 4% and synergist tests) belonged to An. peditaeniatus. The degree of resistance in An. peditaeniatus to each of these three insecticides was approximately 50%. Addition of PBO (Piperonyl butoxide), but not DEF (S.S.S-tributyl phosphotritioate), seemed to restore susceptibility, indicating a potential role of oxidases as a detoxifying enzyme resistance mechanism. Conclusions A better understanding of mosquito diversity related to host preference, biting activity and insecticide resistance status will facilitate the implementation of locally adapted vector control strategies.

Aedes-borne virus disease control relies on insecticides to interrupt transmission. Temephos remains a key chemical for control of immature stage Aedes in Thailand and much of Southeast Asia. However, repeated use of insecticides may result in selection for resistance in vector populations, thus compromising operational intervention. Herein, the phenotypic response to temephos by Aedes aegypti (L.) and Aedes albopictus (Skuse) collected in Thailand and surrounding countries is presented. Data from 345 collection sites are included: 283 from literature review (244 sites with Ae. aegypti, 21 with Ae. albopictus, and 18 having both species sampled), plus 62 locations with Ae. aegypti in Thailand conducted between 2014 and 2018. Susceptibility assays followed WHO guidelines using the recommended discriminating dose of temephos (0.012 mg/liter) against late third to early fourth instar Ae. aegypti. Findings revealed 34 locations with susceptible Ae. aegypti, 13 with suspected resistance, and 15 indicating resistance. Published data between 1999 and 2019 in Thailand found Ae. aegypti resistant in 73 of 206 collection sites, whereas 3 locations from 11 sampled with low-level resistant in Ae. albopictus. From surrounding countries conducting temephos assays (Cambodia, Lao PDR, Myanmar, Malaysia, and Singapore), resistance is present in Ae. aegypti and Ae. albopictus from 27 of 56 and 19 of 28 locations, respectively. Routine insecticide susceptibility monitoring should be an operational requirement in vector control programs. Given the wide distribution and apparent increase in temephos-resistance, alternative larvicidal compounds must be considered if chemical control is to remain a viable vector control strategy.

Monitoring insecticide resistance of Aedes albopictus is required for implementing effective dengue and chikungunya vector control in Thailand. The World Health Organization standard susceptibility test for adult mosquitoes was used to determine the baseline susceptibility of a pyrethroid-susceptible laboratory strain of Ae. albopictus to 5 different pyrethroids (deltamethrin, permethrin, bifenthrin, cypermethrin, and α-cypermethrin). Subsequently, the respective established diagnostic doses (0.026% deltamethrin, 1.024% permethrin, 0.570% bifenthrin, 0.237% cypermethrin, and 0.035% α-cypermethrin) were used to test field-collected Ae. albopictus from Rayong, Koh Chang, and Pong Nom Ron. As expected, the laboratory strain was completely susceptible to all pyrethroid insecticides at the established concentrations. Rayong mosquitoes were found to be highly susceptible to bifenthrin, cypermethrin, and α-cypermethrin. Koh Chang mosquitoes were susceptible to only deltamethrin and permethrin. Pong Nom Ron mosquitoes were resistant to all pyrethroids tested. Routine assessment of these baseline results should guide future resistance monitoring to pyrethroid insecticides in Ae. albopictus in Thailand.

Malaria is a serious mosquito-borne disease transmitted to humans by Anopheles mosquitoes. Seven species of Anopheles are important malaria vectors in Thailand and all remain susceptible to pyrethroid insecticides, despite decades of use in public health mosquito control programs. However, for a more refined and accurate approach to temporal monitoring of susceptibility patterns to commonly used pyrethroids to control adult Anopheles mosquitoes, it is preferred to have specific concentrations for more discriminating testing. A laboratory strain of Anopheles minimus Theobald was used to establish baseline concentrations for susceptibility to six different synthetic pyrethroids-deltamethrin, permethrin, bifenthrin, cypermethrin, α-cypermethrin, and λ-cyhalothrin using the World Health Organization (WHO) adult bioassay system. Final discriminating concentrations of each active ingredient were determined based on doubling LC99 for each chemical to arrive at the following percent concentrations: deltamethrin (0.009%), permethrin (0.551%), bifenthrin (0.12%), cypermethrin (0.104%), α-cypermethrin (0.034%), and λ-cyhalothrin (0.019%). For permethrin, deltamethrin, α-cypermethrin, and λ-cyhalothrin, all revised percentages were lower than currently recommended discriminating concentrations by WHO. Using the newly derived concentrations, insecticide susceptibility tests were followed in a field population of An. minimus s.l. from Kanchanaburi Province. Deltamethrin, bifenthrin, and λ-cyhalothrin produced 100% kill, whereas permethrin, cypermethrin, and α-cypermethrin gave between 92.9 and 97.7% mortality, indicating lower sensitivity (tolerance). Routine monitoring of insecticide susceptibility in An. minimus wild populations throughout Thailand should continue to ensure that insecticides in use are effective to control this vector species.

Malaria is an important public health problem in Thailand, especially along international borders. In this study, we conducted a longitudinal entomological survey in six villages and rubber plantation sites to address the spatio-temporal abundance and behavior of malaria vectors in Ubon Ratchathani Province along the Thailand-Laos border. Adult female mosquitoes were collected by human landing collections (indoor and outdoor) and by cattle bait collections twice per year, during rainy and dry seasons. Mosquitoes were morphologically identified and sibling species were determined by allele-specific PCR. Of the 10,024 Anopheles, 9,328 (93.1%) and 696 (6.9%) were collected during the rainy and dry seasons, respectively. A total of 9,769 (97.5%) and 255 (2.5%) was collected on cattle and human baits, respectively. Very few primary and secondary malaria vectors were collected, consisting of 12 specimens of An. dirus, eight An. minimus, and seven An. aconitus. Of the 152 specimens of the Maculatus Group, only three were identified to An sawadwongporni by molecular methods. The others were 112 An. rampae, a non-vector, that were not amplified or were misidentified as other non-vectors. The very low density of primary malaria vectors found in the study villages suggests that entomological risk and malaria transmission is higher in neighboring forest areas. Further studies on malaria vector distribution, as well as human behaviors, are needed to understand malaria transmission dynamics in the province and to develop suitable vector control methods.

Chemicals can protect humans from the bites of hemophagous arthropods through three different primary actions: irritancy (excitation), repellency, or toxicity, actions that can be evaluated using a laboratory-based assay system. In this study, the deterrent and toxic actions of three synthetic pyrethroids and DDT were characterized on six field strains of Aedes aegypti from Thailand under laboratory-controlled conditions using the high throughput screening system. All six strains showed significant contact irritant responses to the three synthetic pyrethroids, but significantly weaker irritant responses to DDT. Marked repellency responses were seen in all six Ae. aegypti test strains exposed to DDT, whereas the synthetic pyrethroids resulted in greater toxicity than DDT under similar test conditions. Although significantly different in actions, irritancy and repellency may reflect and be influenced by the background insecticide susceptibility status of a particular mosquito population. Results from this study can be used to guide decision making regarding more effective Ae. aegypti adult control in Thailand.

In this study, nine strains of Aedes aegypti mosquitoes from different localities in Thailand were subjected to susceptibility tests against commonly used insecticides for vector control in Thailand. Aedes aegypti from different localities were strongly resistant to DDT and permethrin, except for one strain from Chiang Mai (Chiang Mai-P) that was found to be susceptible to permethrin. In contrast, most Ae. aegypti strains are susceptible to deltamethrin and alphacypermethrin, except that Bangkok and Nonthaburi strains showed incipient resistance to deltamethrin and alphacypermethrin, respectively. Various levels of malathion and propoxur susceptibility were also detected in Ae. aegypti strains; however, two strains of Ae. aegypti (Satun and Nonthaburi) were found to be completely susceptible to malathion. The insecticide susceptibility level in three strains of Culex quinquefasciatus was also investigated. The results indicated strong resistance to DDT and moderate resistance to permethrin. Two strains from Bangkok and Nonthaburi were found to be completely susceptible to malathion and propoxur. One strain from Pathum Thani showed incipient resistance to both compounds. We conclude that deltamethrin and alphacypermethrin are still effective insecticides for the dengue control program and malathion and propoxur may be alternative insecticides to control Cx. quinquefasciatus.

The capacity of mosquitoes to resist insecticides threatens the control of diseases such as dengue and malaria. Until alternative control tools are implemented, characterizing resistance mechanisms is crucial for managing resistance in natural populations. Insecticide biodegradation by detoxification enzymes is a common resistance mechanism; however, the genomic changes underlying this mechanism have rarely been identified, precluding individual resistance genotyping. In particular, the role of copy number variations (CNVs) and polymorphisms of detoxification enzymes have never been investigated at the genome level, although they can represent robust markers of metabolic resistance. In this context, we combined target enrichment with high-throughput sequencing for conducting the first comprehensive screening of gene amplifications and polymorphisms associated with insecticide resistance in mosquitoes. More than 760 candidate genes were captured and deep sequenced in several populations of the dengue mosquito Ae. aegypti displaying distinct genetic backgrounds and contrasted resistance levels to the insecticide deltamethrin. CNV analysis identified 41 gene amplifications associated with resistance, most affecting cytochrome P450s overtranscribed in resistant populations. Polymorphism analysis detected more than 30,000 variants and strong selection footprints in specific genomic regions. Combining Bayesian and allele frequency filtering approaches identified 55 nonsynonymous variants strongly associated with resistance. Both CNVs and polymorphisms were conserved within regions but differed across continents, confirming that genomic changes underlying metabolic resistance to insecticides are not universal. By identifying novel DNA markers of insecticide resistance, this study opens the way for tracking down metabolic changes developed by mosquitoes to resist insecticides within and among populations.

The objective of this study was to compare the behavioral responses (contact irritancy and noncontact spatial repellency) between susceptible and resistant populations of Aedes aegypti (L.) (=Stegomyia aegypti) to essential oils, citronella, and eucalyptus, Eucalyptus globulus, extracts, using an excito-repellency test system. N, N-diethyl-meta-toluamide (DEET) was used as the standard reference repellent. Mosquitoes included two long-standing insecticide susceptible colonies (U.S. Department of Agriculture and Bora Bora) and two pyrethroid-resistant populations recently obtained from Phetchabun and Kanchanaburi provinces in Thailand. Both DEET and citronella produced a much stronger excitation ("irritancy") and more rapid flight escape response in both pyrethroid-resistant populations compared with the laboratory populations. Noncontact repellency was also greater in the two resistant populations. Eucalyptus oil was found to be the least effective compound tested. Differences in responses between long-established pyrethroid-susceptible colonies and newly established and naturally resistant colonies were clearly demonstrated. These findings also demonstrate the need for further comparisons using natural pyrethroid-susceptible populations for elucidation of factors that might contribute to different patterns of escape behavior.

Establishing baseline insecticide discriminating doses is crucial in accurately determining susceptibility status and changing temporal patterns of physiological response in mosquito populations. Pyrethroids are the predominant chemicals used for controlling adult Aedes aegypti and Ae. albopictus, both vectors of dengue viruses, in Thailand. Presently, only 2 pyrethroids, permethrin and lambda-cyhalothrin, have published diagnostic dose rates for monitoring Ae. aegypti. This study established the diagnostic lethal concentrations for 6 different pyrethroids available in Thailand for dengue vector control. United States Department of Agriculture insecticide-susceptible strain of Ae. aegypti was used to establish the baseline concentrations for subsequent susceptibility testing of field populations. Our findings showed lower discriminating concentrations for lambda-cyhalothrin and permethrin than those recommended by the World Health Organization (WHO), at 2.5- and 1.7-fold lower dosing, respectively. The susceptibility status of 3 different geographical populations of field-collected Ae. aegypti were tested using the standard WHO procedures. All 3 field strains demonstrated varying levels of physiological resistance to each compound. We conclude that establishing the baseline diagnostic concentration of an insecticide is of paramount importance in accurately determining the susceptibility status in field-collected mosquitoes. If possible, discriminating doses should be established for all insecticides and test assays run concurrently with a known susceptible strain for more accurate monitoring of resistance in mosquito populations in Thailand.

This study quantified both contact irritancy and noncontact repellency behavioral responses of three strains of Aedes aegypti (L.) (one long-term colony and two F1-F2 generation field-caught strains) to field application rates of DDT (2 g/m2) and alpha-cypermethrin (ACyp) (0.025 g/m2) by using an excito-repellency test chamber. The colony The colony strain (USDA) was completely susceptible to DDT and ACyp. One field strain was collected from Chiang Mai (CM) Province, northern Thailand, and was characterized as tolerant (reduced susceptibility) to DDT and completely susceptible to ACyp. The second field strain, collected from Kanchanaburi (KAN) Province, western Thailand, was highly resistant to DDT but fully susceptible to ACyp. All three strains exhibited marked irritancy to contact with ACyp, with more pronounced escape responses occurring in the two field strains. With DDT, the KAN strain demonstrated the lowest escape response during both contact and noncontact trials, whereas a greater response was seen in trials conducted with CM and USDA strains. With exposure to ACyp, repellency was less profound than irritancy but still resulted in a significant escape response compared with paired controls without insecticide (P0.05). DDT elicited both irritancy and repellency responses but comparably greater spatial repellency than ACyp. Findings indicate ACyp functions primarily as a strong contact irritant, whereas DDT functions as a relatively strong noncontact repellent in the strains tested. The higher the degree of physiological resistance to DDT, the greater the apparent suppression of both behavioral avoidance responses. Most importantly, observations using susceptible, tolerant, and resistant Ae. aegypti strains show that behavioral responses that can interrupt human-vector contact still occur regardless of degree of physiological susceptibility to compounds tested.