Comparing Light-Emitting-Diodes Light Traps for Catching Anopheles Mosquitoes in a Forest Setting, Western Thailand.

Light traps are a common method for attracting and collecting arthropods, including disease vectors such as mosquitoes. Various types of traps have been used to monitor mosquitoes in a forest in Western Thailand. In this study, four Light Emitting Diodes (LED) light sources (UV, blue, green, and red) and two fluorescent lights (white and UV) were used to trap nocturnal adult mosquitoes. These traps were used with light alone and not any additional attractant. The experiment was conducted from 18:00 to 06:00 h. on six consecutive nights, every two months, across dry, wet, and cold seasons. All specimens were first identified by morphological features and subsequently confirmed by using PCR. We collected a total of 873 specimens of 31 species in four genera, Anopheles, Aedes, Culex, and Armigeres. Anopheles harrisoni was the predominant species, followed by Aedes albopictus, Culex brevipalpis, Culex nitropunctatus, and Armigeres (Leicesteria) longipalpis. UV fluorescent light was the most effective light source for capturing forest mosquitoes, followed by UV LED, blue LED, green LED, white fluorescent, and red LED. The optimal times for collection were from 21:00 to 03:00 h in the dry season. Our results demonstrate that appropriate sampling times and light sources should be selected for optimal efficiency in vector surveillance programs.

Excito-repellent activity of ß-caryophyllene oxide against Aedes aegypti and Anopheles minimus.

Contact irritant and non-contact repellent activities of ß-caryophyllene oxide were evaluated against laboratory strains of female Aedes aegypti (USDA strain), a major arbovirus vector and Anopheles minimus (KU strain), a major malaria parasite vector, compared with the synthetic repellent DEET, using an excito-repellency test system. ß-caryophyllene oxide and DEET were tested at concentrations of 0.1, 0.25, 0.5 and 1.0% (v/v). Anopheles minimus was found to be more sensitive to ß-caryophyllene oxide than that of Ae. aegypti and exhibited high avoidance response rates (86-96% escape) at 0.5% and 1.0% concentrations in contact and non-contact trials compared with Ae. aegypti (22-59% escape). However, at the same concentrations, DEET displayed lower irritancy and repellency capacities against these two mosquito species (range 0-54% escape) compared to ß-caryophyllene oxide. The analysis of escape responses showed significant differences between mosquito species at all concentrations (P < 0.05) except for 0.1%. For both species, there were significant differences in irritant and repellent responses between ß-caryophyllene oxide and DEET at higher concentrations (0.5 and 1.0%).

Enhanced mortality in deltamethrin-resistant Aedes aegypti in Thailand using a piperonyl butoxide synergist.

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 < 0.05) in final 24-h mortality, except marginally for Lampang (P = 0.053). The synergistic effects of PBO with deltamethrin-resistant Ae. aegypti suggest a combination of this synergist with deltamethrin or other pyrethroid compounds can significantly enhance the effectiveness of these insecticides against pyrethroid-resistant Ae. aegypti found commonly in Thailand.

Excito-Repellency of Citrus hystrix DC Leaf and Peel Essential Oils Against Aedes aegypti and Anopheles minimus (Diptera: Culicidae), Vectors of Human Pathogens.

The essential oils of kaffir lime (Citrus hystrix DC.) at four different concentrations (0.5, 1.0, 2.5, and 5.0% v/v) were studied for their repellency, excitation, and knockdown properties against laboratory strains of Aedes aegypti (L.) and Anopheles minimus Theobald using an excito-repellency test system. Both contact and noncontact escape responses to leaf- and peel-derived kaffir lime oils were observed. Comparing unadjusted escape responses for An. minimus, leaf oil had strong combined irritant and repellent activity responses at 1-5% concentrations (90.0-96.4% escape) and the strongest spatial repellent activity at 1% and 2% (85.9% and 87.2% escape, respectively). The peel oil exhibited good excitation with repellency at concentrations of 2.5% (89.8% escape) and 5% (96.28% escape), while concentrations 1-5% showed more moderate repellent activity against An. minimus. For Ae. aegypti, 2.5% leaf oil produced the greatest response for both contact (56.1% escape) and noncontact (63.3% escape) trials, while 2.5% produced the strongest response among all concentrations of peel oil, with 46.5% escape. However, after adjusting the contact trial escape (a measure of combined excitation and repellency), the estimated escape due to contact alone was a much weaker response than spatial repellency for both species. Knockdown responses above 50% were only observed in Ae. aegypti exposed to 5% leaf oil. Kaffir lime oils were more active against An. minimus than Ae. aegypti mosquitoes. There were statistically significant differences between leaf (more active) and peel oils at each concentration against An. minimus in contact and noncontact trials, except at the highest (5%) concentration.

Evaluation of a Noncontact, Alternative Mosquito Repellent Assay System.

A novel noncontact repellency assay system (NCRAS) was designed and evaluated as a possible alternative method for testing compounds that repel or inhibit mosquitoes from blood feeding. Deet and Aedes aegypti were used in a controlled laboratory setting. Using 2 study designs, a highly significant difference were seen between deet-treated and untreated skin placed behind the protective screens, indicating that deet was detected and was acting as a deterrence to mosquito landing and probing behavior. However, a 2nd study showed significant differences between protected (behind a metal screen barrier) and unprotected (exposed) deet-treated forearms, indicating the screen mesh might restrict the detection of deet and thus influences landing/biting response. These findings indicate the prototype NCRAS shows good promise but requires further evaluation and possible modification in design and testing protocol to achieve more desirable operational attributes in comparison with direct skin-contact repellency mosquito assays.

Comparison of Field and Laboratory-Based Tests for Behavioral Response of Aedes aegypti (Diptera: Culicidae) to Repellents.

The repellent and irritant effects of three essential oils-clove, hairy basil, and sweet basil-were compared using an excito-repellency test system against an insecticide-resistant strain of Aedes aegypti (L.) females from Pu Teuy, Kanchanaburi Province. DEET was used as the comparison standard compound. Tests were conducted under field and controlled laboratory conditions. The most marked repellent effect (spatial noncontact assay) among the three test essential oils was exhibited by sweet basil, Ocimum basilicum L. (53.8% escaped mosquitoes in 30-min exposure period) under laboratory conditions while hairy basil, Ocimum americanum L. and clove, Syzygium aromaticum (L.) Merill et. L.M. Perry from laboratory tests and sweet basil from field tests were the least effective as repellents (0-14%). In contrast, the contact assays measuring combined irritancy (excitation) and repellency effects found the best contact irritant response to hairy basil and DEET in field tests, whereas all others in laboratory and field were relatively ineffective in stimulating mosquitoes to move out the test chambers (0-5.5%). All three essential oils demonstrated significant differences in behavioral responses between field and laboratory conditions, whereas there was no significant difference in contact and noncontact assays for DEET between the two test conditions (P > 0.05).

Anopheles species diversity and distribution of the malaria vectors of Thailand.

In Thailand, seven Anopheles species within three species assemblages have been incriminated as important malaria vectors. Comprehensive maps showing the countrywide geographical distribution of primary and secondary malaria vector species have not yet been developed; the maps that do exist are typically restricted to specific areas or are out of date. In addition, with the advent of molecular-based species-identification tools, the geographical locations of various sibling species have been more clearly defined in the country. This review has compiled published data on the known distribution of important malaria vectors in Thailand based on genetic and molecular identification assays from 1994 to 2014, with maps of relevant species distribution provided herein.

Review of insecticide resistance and behavioral avoidance of vectors of human diseases in Thailand.

Physiological resistance and behavioral responses of mosquito vectors to insecticides are critical aspects of the chemical-based disease control equation. The complex interaction between lethal, sub-lethal and excitation/repellent ('excito-repellent') properties of chemicals is typically overlooked in vector management and control programs. The development of "physiological" resistance, metabolic and/or target site modifications, to insecticides has been well documented in many insect groups and disease vectors around the world. In Thailand, resistance in many mosquito populations has developed to all three classes of insecticidal active ingredients currently used for vector control with a majority being synthetic-derived pyrethroids. Evidence of low-grade insecticide resistance requires immediate countermeasures to mitigate further intensification and spread of the genetic mechanisms responsible for resistance. This can take the form of rotation of a different class of chemical, addition of a synergist, mixtures of chemicals or concurrent mosaic application of different classes of chemicals. From the gathered evidence, the distribution and degree of physiological resistance has been restricted in specific areas of Thailand in spite of long-term use of chemicals to control insect pests and disease vectors throughout the country. Most surprisingly, there have been no reported cases of pyrethroid resistance in anopheline populations in the country from 2000 to 2011. The precise reasons for this are unclear but we assume that behavioral avoidance to insecticides may play a significant role in reducing the selection pressure and thus occurrence and spread of insecticide resistance. The review herein provides information regarding the status of physiological resistance and behavioral avoidance of the primary mosquito vectors of human diseases to insecticides in Thailand from 2000 to 2011.

Challenges and prospects for dengue and malaria control in Thailand, Southeast Asia.

Despite significant advances in the search for potential dengue vaccines and new therapeutic schemes for malaria, the control of these diseases remains difficult. In Thailand, malaria incidence is falling whereas that of dengue is rising, with an increase in the proportion of reported severe cases. In the absence of antiviral therapeutic options for acute dengue, appropriate case management reduces mortality. However, the interruption of transmission still relies on vector control measures that are currently insufficient to curtail the cycle of epidemics. Drug resistance in malaria parasites is increasing, compromising malaria control and elimination. Deficiencies in our knowledge of vector biology and vectorial capacity also hinder public health efforts for vector control. Challenges to dengue and malaria control are discussed, and research priorities identified.

Seasonal abundance and distribution of Anopheles larvae in a riparian malaria endemic area of western Thailand.

Three taxonomic groups of Anopheles larvae were morphologically identified within the Funestus Group (Minimus Subgroup and Aconitus Subgroup) (75.63%), Maculatus Group (20.47%), and Barbirostris Group (0.57%) during a two-year period in conjunction with active malaria transmission in a village near the Thai-Myanmar border in Kanchanaburi Province, western Thailand. The remaining 3.33% of anophelines collected were Anopheles culicifacies (3.07%), Anopheles philippinensis (0.17%), and Anopheles vagus (0.09%). Using an allele-specific multiplex molecular identification assay, the Minimus Subgroup consisted of Anopheles minimus (69.83%), and Anopheles harrisoni (0.06%) and 2 genetically-related species belonging to the Aconitus Subgroup, Anopheles aconitus (0.63%) and Anopheles varuna (5.12%). The Minimus and Aconitus Subgroup species were more abundant during the dry season (52.58%) than during the hot (24.95%) and wet (22.46%) seasons. The number of Anopheles larvae collected from the stream habitat was significantly higher during the second year than the first year, believed to be due to human environmental changes in the stream habitat from the building of a small check dam, which provided a more suitable and stable habitat for mosquito larval development. This study illustrates the importance of conducting site-specific studies to accurately determine vector bionomics (eg, larval habitats) and adult activity patterns and linking observations with malaria transmission dynamics in a given area.

Irritant and repellent responses of Anopheles harrisoni and Anopheles minimus upon exposure to bifenthrin or deltamethrin using an excito-repellency system and a live host.

Feeding responses of Anopheles harrisoni and An. minimus were evaluated following exposure to 2 pyrethroid insecticides, bifenthrin or deltamethrin, using an excito-repellency test system in the presence and absence of live host cues. The results demonstrated that contact irritancy was the primary action of bifenthrin or deltamethrin in both mosquito species. There was no noncontact repellency effect elicited by either insecticide. Anopheles minimus showed rapid escape response with high mortality rates following direct contact with deltamethrin in the absence of a host and delayed escape responses when a host was present. Similarly, exposure of An. minimus to bifenthrin also elicited a delayed escape response in the presence of a host but with lower mortality rates. In experiments using An. harrisoni, the presence or absence of a host had no significant effect on behavioral responses to either insecticide (P > 0.05). We conclude that deltamethrin elicited stronger irritant chemical effects than bifenthrin but that behavioral responses in vector populations are dampened in the presence of an available host. This information is useful for estimating probability of pathogen transmission when using irritant chemicals in proximity to a blood-meal source.

Host feeding responses of Aedes aegypti (L.) exposed to deltamethrin.

Escape responses of mated and unmated nulliparous Aedes aegypti mosquitoes were compared using three different concentrations of deltamethrin in the presence or absence of a live animal host using an excito-repellency (ER) test system. Both insecticide contact (excitation) and non-contact (repellency) test configurations were compared. For contact trials, mated mosquitoes showed similar escape movements among the three concentrations when host stimuli were absent. Significant differences in responses were seen between the lower concentrations of (LC(50) and LC(75) ) deltamethrin with and without hosts present (P<0.05). Presence or absence of host stimuli produced no significant differences in escape response for unmated females when exposed to the highest concentration (LC(90) ) of deltamethrin. Our findings indicate that as deltamethrin concentrations decrease to sublethal levels, mating status and host cues play a more significant role in escape behavior. Therefore, insemination can influence the outcome of feeding success and flight movement of nulliparous female Ae. aegypti in contact with deltamethrin and in the presence of live host stimuli. The ER assay system serves as a useful tool for observing excitation and repellency responses of Ae. aegypti to insecticides in the presence or absence of other environmental and biological cues that can affect mosquito behavior.

Seasonal abundance and bloodfeeding activity of Anopheles dirus sensu lato in western Thailand.

Bloodfeeding activity, host preference, and seasonal abundance of the Anopheles dirus complex in relation to environmental factors were measured during a 2-yr period at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, western Thailand. Anopheles dirus s.l. was found more abundant during the wet season compared with the dry and hot seasons. Bloodfeeding by An. dirus s.l. commenced immediately after sunset with a distinct peak of activity at 2000 h. Anopheles dirus s.l. fed more readily on cattle than on humans, demonstrating a significant but slightly stronger zoophilic behavior (P = 0.02). The biting activity of An. dirus s.l. in Pu Teuy appears to differ from other localities in Thailand, suggesting the existence of significant biological variability within the species complex depending on the local circumstances.

Biting activity and host preference of the malaria vectors Anopheles maculatus and Anopheles sawadwongporni (Diptera: Culicidae) in Thailand.

Adult mosquitoes in the Anopheles maculatus group were surveyed from different regions of Thailand and five different species were morphologically identified, including Anopheles maculatus, Anopheles sawadwongporni, Anopheles notanandai, Anopheles dravidicus, and Anopheles willmori. Blood-feeding activity and host preference of two species, Anopheles maculatus and Anopheles sawadwongporni, were observed during a one-year period at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, west-central Thailand. Both species were more prevalent during the wetter period of the year and each had a greater predilection to feed on cattle than humans. Primary feeding activity occurred between 20:00-23:00 and a smaller peak at 01:00-03:00. Findings are discussed relative to the importance of these two vectors for malaria transmission in Pu Teuy.

Influence of nutritional and physiological status on behavioral responses of Aedes aegypti (Diptera: Culicidae) to deltamethrin and cypermethrin.

Excito-repellency responses of Aedes aegypti (L.) exposed to deltamethrin and cypermethrin were assessed using an excito-repellency test system. Contact irritancy and non-contact repellency assays compared non-bloodfed (unfed) parous (post-gravid), nulliparous, early blood-fed, late blood-fed, sugar-fed, and unmated female mosquitoes for behavioral responses based on nutritional and physiological conditions at the time of testing. Rates of escape during contact exposure with either compound were most pronounced in parous mosquitoes, followed by unmated mosquitoes, when compared to other conditional states. Significantly higher numbers of parous females also escaped from control chambers compared to other cohorts (P <0.05). Irritability of blood- and sugar-fed mosquitoes was noticeably suppressed. We conclude that nutritional and physiological conditions (including age) of mosquitoes at the time of testing can significantly influence behavioral responses (excito-repellency) to insecticides. The findings indicate that whether due to chronological age, nutrition, physiological state, or innate (circadian) activity patterns, careful consideration must be given to the selection of appropriate conditioned mosquitoes for testing.

Behavioral responses of Aedes aegypti (Diptera: Culicidae) exposed to deltamethrin and possible implications for disease control.

Behavioral responses of nine Aedes aegypti (L.) strains, six from recent field collections and three from the long-established laboratory colonies, were tested under laboratory-controlled conditions by using an excito-repellency test system. All nine strains showed significant behavioral escape responses when exposed to deltamethrin at the standard field dose (0.02 g/m2), regardless of background insecticide susceptibility status (susceptible or tolerant/resistant). Insecticide contact irritancy played a predominate role in overall female mosquito escape responses, whereas noncontact repellency was not observed at levels significantly different from paired noncontact control tests (P > 0.01). Among the six field populations, the Jakarta (Indonesia) Toba (north Sumatra), and Bangkok female mosquitoes showed rapid exit (>78%) during 30 min of direct contact with insecticide-treated surfaces, whereas the other three strains demonstrated only moderate escape responses (32-56%) from the chambers. Moderate escape responses during direct insecticidal contact also were observed in the three laboratory test populations (44- 60%). Higher percentage of mortality was observed from laboratory strains (8-33%) that failed to escape compared with nonescape females of field strains (2-16%), possibly a reflection of background deltamethrin susceptibility status. We conclude that contact irritancy is a major behavioral response of Ae. aegypti when exposed directly to deltamethrin and that rapid flight escape from areas exposed to space sprays or surfaces treated with residual pyrethroids could have a significant impact on the effectiveness of adult mosquito control and disease transmission reduction measures.