Irritant and repellent behaviors of sterile male Aedes aegypti (L.) (Diptera: Culicidae) mosquitoes are crucial in the development of disease control strategies applying sterile insect technique.

The mosquito Aedes aegypti, known to transmit important arboviral diseases, including dengue, chikungunya, Zika and yellow fever. Given the importance of this disease vector, a number of control programs have been proposed involving the use of the sterile insect technique (SIT). However, the success of this technique hinges on having a good understanding of the biology and behavior of the male mosquito. Behavioral responses of Ae. aegypti male populations developed for SIT technology were tested under laboratory conditions against chemical and natural irritants and repellents using an excito-repellency (ER) chamber. The results showed that there were no significant behavioral escape responses in any of the radiation-sterilized male Ae. aegypti test populations when exposed to citronella, DEET, transfluthrin, and deltamethrin, suggesting that SIT did not suppress the expected irritancy and repellency (avoidance) behaviors. The type of information reported in the current study is vital in defining the effects of SIT on vector behavior and understanding how such behavior may influence the success of SIT technology with regard to other vector control interventions.

Effects of mosquito age and batch size on knockdown and mortality of laboratory-reared Anopheles dirus, Anopheles minimus, and wild-caught Anopheles harrisoni (Diptera: Culicidae) exposed to transfluthrin using WHO tube and CDC bottle bioassays.

WHO tube and CDC bottle bioassays are currently available for insecticide resistance monitoring and malaria transmission research. Multiple parameters including mosquito density, age, and nutritional status may affect the readout in these bioassays' tests. This study aims to assess the effects of experimental factors on knockdown and mortality measurements in dominant malaria vectors in Thailand following exposure to sublethal and lethal doses of transfluthrin. The effects of (i) 3 different mosquito batch sizes (5, 10, and 20 individuals) and (ii) 2 age groups (3-5 and 20-23 days old) on outcomes measured using the WHO tube (14.7 µg/cm2) and CDC bottle bioassay discriminating concentration (0.006 µg/cm2) against 2 laboratory strains: Anopheles dirus Peyton & Harrison and Anopheles minimus Theobald (species A) and wild-caught Anopheles harrisoni Harbach & Manguin (species C). Our results showed higher knockdown at 1-h exposure using WHO tube and CDC bottle bioassays containing 20 individuals compared to batches containing 10 and 5 individuals. Older mosquitoes showed greater susceptibility than younger test population, especially for An. mininus. Our study supports WHO recommendations for using 3- to 5-day-old mosquitoes. It also validates Praulin et al. (2022) proposal to divide the cohort into smaller batches with more test replicates when it is not practicable to test 25 mosquitoes per replicate.

Synergistic Repellent and Irritant Effects of a Mixture of ß-Caryophyllene Oxide and Vetiver Oil against Mosquito Vectors.

Repellents play a major role in reducing the risk of mosquito-borne diseases by preventing mosquito bites. The present study evaluated the mosquito-repellent activity of ß-caryophyllene oxide 1% (BCO), vetiver oil 2.5% (VO), and their binary mixtures (BCO + VO (1:1), BCO + VO (2:1), BCO + VO (1:2)) against four laboratory-colonized mosquito species, Aedes aegypti (L.), Aedes albopictus (Skuse), Anopheles minimus Theobald, and Culex quinquefasciatus Say, using an excito-repellency assay system. In general, the compound mixtures produced a much stronger response in the mosquitoes than single compounds, regardless of the test conditions or species. The greatest synergetic effect was achieved with the combination of BCO + VO (1:2) in both contact and noncontact trials with An. minimus (74.07-78.18%) and Cx. quinquefasciatus (55.36-83.64%). Knockdown responses to the binary mixture of BCO + VO were observed for Ae. albopictus, An. minimus, and Cx. quinquefasciatus, in the range of 18.18-33.33%. The synergistic repellent activity of BCO and VO used in this study may support increased opportunities to develop safer alternatives to synthetic repellents for personal protection against mosquitoes.

Synergistic Behavioral Response Effect of Mixtures of Andrographis paniculata, Cananga odorata, and Vetiveria zizanioides against Aedes aegypti (Diptera: Culicidae).

Each binary mixture formulation of Vetiveria zizanioides (L.) Nash (VZ) with Andrographis paniculata (Burm.f.) Wall. ex Nees (AP) or Cananga odorata (Lam.) Hook.f. & Thomson (CO) and AP with CO at 1:1, 1:2, 1:3, and 1:4 ratios (v:v) was investigated for behavioral responses on laboratory and field strains of Aedes aegypti. Irritant and repellent activities of each formulation were compared with N,N-diethyl-3-methylbenzamide (DEET) using an excito-repellency test system. The result demonstrated that the mixture of VZ:AP in all combination ratios was the most effective in inducing an irritancy response against the laboratory strain (56.57-73.33%). The highest percentage of escaped mosquitoes exposed to the mixture at a 1:4 ratio (73.33%) was significantly different from DEET (26.67%) (p < 0.05). Against the field strain, the strongest escape response of AP:CO at a 1:1 ratio in the contact trial (70.18%) was significantly different compared with DEET (38.33%) (p < 0.05). There was a weak non-contact escape pattern in all combinations of VZ:CO against the laboratory strains (6.67-31.67%). These findings could lead to the further development of VZ and AP as active ingredients in a repellent that could advance to human use trials.

Enhanced Excito-Repellency of Binary Mixtures of Plant-Based Mosquito Repellents Against Culex quinquefasciatus Say (Diptera: Culicidae), a Night Biting Mosquito Species.

Efficacies of essential oils (EOs) of Vetiveria zizanioides (L.) Nash. (Poales: Poaceae) (VZ EO), Cananga odorata (Lam) Hook. F. & Thomson (Magnoliales: Annonaceae) (CO EO), and crude extract (CE) of Andrographis paniculata (Burm.F.) Wall ex. Nees (Lamiales: Acanthaceae) (AP CE), against laboratory (lab) and field strains of Culex quinquefasciatus Say were investigated. Irritant and repellent activities of individual and binary mixtures of plant extracts were compared with N,N-diethyl-m-toluamide (DEET) using an excito-repellency system. The irritant activity (direct tarsal contact), the mean percent escape response of VZ EO (91.67%, 83.33%), and CO EO (80%, 88.33%) were not significantly different compared with DEET (88.33%, 95%) against lab and field strains, respectively. Similarly, irritant responses in combinations (1:1 and 1:2, v:v) of either VZ EO or CO EO with AP CE were not significantly different from DEET against both strains (P > 0.001). The repellent activity (no tarsal contact), the mean percent escape response of VZ EO (68.33%), CO EO (61.67%), and VZ EO+AP CE (1:1, v:v) (81.67%) against lab strain and CO EO (85%) against field strain were not significantly different from that of DEET (P > 0.001). Interestingly, the greatest contact irritancy of VZ EO+AP CE (1:1, v:v) (96.67%) (P = 0.0026) and a stronger repellency response of CO EO (85%) (P = 0.0055) produced significantly different patterns of escape response compared with DEET against both lab and field strains, respectively. The EOs of VZ EO and CO EO or their mixture with AP CE showed potential as plant-based active ingredients for mosquito repellents. In addition, the major chemical constituents of VZ EO were ß-vetivone (6.4%), khusimol (2.96%), and α-vetivone (2.94%) by gas chromatograpy-mass spectrometry.

Behavioral avoidance and biological safety of vetiver oil and its constituents against Aedes aegypti (L.), Aedes albopictus (Skuse) and Culex quinquefasciatus Say.

Numerous plant-based repellents are widely used for personal protection against host-seeking mosquitoes. Vitiveria zizanioides (L.) Nash essential oil and its constituents have demonstrated various mosquito repellent activities. In this study, three chemical actions of vetiver oil and five constituents (terpinen-4-ol, α-terpineol, valencene, vetiverol and vetivone) were characterized against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus by using the high-throughput screening assay system (HITSS). Significant contact escape responses in Ae. aegypti and Ae. albopictus to all test compounds at concentrations between 2.5 and 5% were observed. Spatial repellency responses were also observed in some tested mosquito populations depending upon concentrations. The most significant toxic response on mosquitoes was found at the highest concentration, except for vetivone which had no toxic effect on Ae. aegypti and Ae. albopictus. Results on phototoxic and genotoxic hazard revealed that vetiver oil and their constituents showed no phototoxic potential or any significant genotoxic response. In conclusion, vetiver oil and two constituents, valencene and vetiverol, are potentials as active ingredients for mosquito repellency and present no toxicity.

Cananga odorata (Magnoliales: Annonaceae) Essential Oil Produces Significant Avoidance Behavior in Mosquitoes.

Essential oil of Cananga odorata Hook. F. & Tomson is a source of insect repellent, but contact irritancy and noncontact repellency actions that stimulate insect's avoidance behavior (escape away from chemical source after direct physical contact or without making physical contact, respectively) have not been investigated. Therefore, an excito-repellency test chamber was used for measuring avoidance behavior of four insectary-reared mosquito species (Diptera: Culicidae) that escape from esposure to four concentrations (0.5, 1.0, 2.5, and 5.0% v/v) of C. odorata oil. The oil strongly repelled both Culex quinquefasciatus Say (85-97% escape) and Anopheles minimus Theobald (97-99%) at high concentrations (2.5-5.0%). For Anopheles dirus Peyton & Harrison and Aedes aegypti (L.), highest repellency (64 and 39% escape, respectively) was demonstrated at 2.5% concentration. For contact irritancy, the oil produced relatively high percent escape found in Cx. quinquefasciatus (90-100% escape) and An. minimus (83-100%). Whereas moderate contact irritancy was observed against An. dirus (40-50% escape) and Ae. aegypti (51-59%). The percent escape was then adjusted with repellency to estimate the effect of contact irritancy alone. We found that highest contact irritancy was presented at 0.5% concentration against An. minimus (67% escape). Knockdown and toxic actions were only found in Anopheles mosquitoes at 5.0% concentration. The results revealed that An. minimus and Cx. quinquefasciatus were more prone to be repelled by C. odorata oil. Detailed analysis of oil identified primary compounds as methyl benzoate (14.6%), α-gurjunene (12.8%), p-methyl-anisole (11.3%), and benzyl acetate (9.9%). Further investigations are needed to assess excito-repellency actions of these compounds alone or in combination.

Behavioral responses to transfluthrin by Aedes aegypti, Anopheles minimus, Anopheles harrisoni, and Anopheles dirus (Diptera: Culicidae).

Airborne spatial repellency (SR) is characterized and distinguished from other chemical actions including contact locomotor excitation and toxicity. The use of volatile spatial repellents is a potential new intervention class for combatting mosquito-borne pathogen transmission; therefore, continuing investigations on the actions of these chemicals that modify mosquito host-seeking behavior (i.e., bite prevention) is needed. The objective of this study is to characterize the key behavioral avoidance actions of transfluthrin (TFT) to advance spatial repellent development into practical products. Behavioral avoidance responses were observed for adult laboratory strains of Aedes aegypti, Anopheles minimus and An. dirus, and two field populations of An. harrisoni and Ae. aegypti, respectively. Established TFT sublethal (LC50 and LC75), lethal concentrations (LC99) and discriminating concentrations (DCs) were selected corresponding to each mosquito test species. Spatial repellency and contact excitation ('irritancy') responses on adult mosquitoes to TFT were assessed using an excito-repellency assay system. At LC50, TFT exhibited strong avoidance with An. minimus (60.1% escape) and An. dirus (80% escape) laboratory strains, showing between 12 and 16x greater escape response than Ae. aegypti (5% escape). Repellency responses for field collected Ae. aegypti and An. harrisoni were 54.9 and 47.1% escape, respectively. After adjusting the initial contact escape response (a measure of combined irritancy and repellency) to estimate only escape due to contact, the LC50 and LC99 showed moderate escape irritancy with laboratory Ae. aegypti (41.4% escape) and no contact activity against the field population. Adjustment showed only weak contact activity (16.1% escape) in laboratory An. minimus at LC50. Spatial repellency is the predominant mode of action of TFT among colonized and field mosquitoes used in this study. Established baseline (susceptible) dose-response curves assist in optimizing SR products for mosquito control and pathogen transmission prevention.

Excito-repellency and biological safety of ß-caryophyllene oxide against Aedes albopictus and Anopheles dirus (Diptera: Culicidae).

The activity of ß-caryophyllene oxide as either a contact or noncontact repellent was evaluated against two laboratory strains (Aedes albopictus and Anopheles dirus) using an excito-repellency test system. N, N-Diethyl-3-methylbenzamide (DEET) was used as a standard reference baseline for comparative purposes. ß-Caryophyllene oxide and DEET were tested at concentrations of 0.1, 0.25, 0.5 and 1.0% (v/v). In addition, the phototoxic and genotoxic effects of ß-caryophyllene oxide were investigated on Balb/c 3T3 mouse fibroblasts (3T3-L1) and Chinese hamster ovary cell line (CHO-K1). The results demonstrated that the higher concentrations of test compounds (0.5 and 1.0%) produced greater behavioral responses. Aedes albopictus was more sensitive to ß-caryophyllene oxide than An. dirus. Moderate avoidance response rates (25-56% escape) of Ae. albopictus at 0.5% and 1.0% ß-caryophyllene oxide were observed in contact and noncontact trials compared with low response rates from An. dirus (26-31% escape). DEET at ≤1% displayed lower irritancy and repellency (1-38%) than ß-caryophyllene oxide when tested against the two mosquito species. Knockdown responses (37%) were only observed in An. dirus exposed to 1% ß-caryophyllene oxide in the contact trial. ß-Caryophyllene oxide did not show any phototoxic potential (PIF= 0.38) nor was there any significant genotoxic response as indicated by no increase in micro-nucleated cells with or without metabolic activation. ß-Caryophyllene oxide could be considered as a safe repellent, effective against mosquitoes.

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%).

Discriminating Lethal Concentrations for Transfluthrin, a Volatile Pyrethroid Compound for Mosquito Control in Thailand.

Pyrethroids are commonly used to control malaria and dengue vectors in Thailand. The lack of specific lethal discriminating concentrations (DCs) for specific mosquito species has possibly compromised more accurate assessments of physiological susceptibility to various chemicals over time. Previous studies have established DCs of various residual pyrethroids against specific mosquitoes in Thailand. However, DCs for transfluthrin (TFT), a highly volatile pyrethroid compound, against mosquito vectors in Thailand has been lacking. The aim of this study was to determine the DCs and susceptibility baselines of TFT against pyrethroid-susceptible laboratory strains of Aedes aegypti, Anopheles minimus, and An. dirus using the World Health Organization adult susceptibility tube method. Final DCs of TFT of each species were determined based on doubling the 99% lethal concentration at the following percentages: Ae. aegypti (0.06824%), An. minimus (0.06382%), and An. dirus (0.01508%). Subsequently, the respective TFT DCs were used to test field-collected populations of Ae. aegypti, An. harrisoni (Minimus Complex species), and An. dirus. Anopheles harrisoni and An. dirus were found completely susceptible (100% mortality), whereas Ae. aegypti from Nonthaburi Province was resistant to TFT. The suitability of the testing system and procedures is discussed. Routine assessment of insecticide susceptibility should include pyrethroids with high-vapor-pressure characteristics for informing control programs and consumers of product and chemical effectiveness.

Diversity and biting patterns of Anopheles species in a malaria endemic area, Umphang Valley, Tak Province, western Thailand.

Malaria is highly endemic in Umphang Valley, a district in the western edge of Tak Province, along the boundary with Kayin State of Myanmar. Although there are high indigenous malaria cases in this area every year, nothing about malaria vectors and their transmission role have been investigated before this study. The objective of this work is to characterize the Anopheles species diversity and trophic behavior of malaria vectors in the transmission area of Umphang Valley. Females of Anopheles mosquitoes were collected every two months during a two-year period. Mosquito collections were using standard collection technique, indoor and outdoor human landing collections and outdoor cattle bait collection. Anopheles mosquitoes were identified using morphological characters and multiplex AS-PCR assay for the identification of sibling species within groups and complexes present. From a total of 16,468 Anopheles females, 2723 specimens (16.54%) were collected from humans and 13,745 specimens (83.46%) were captured from cattle. From human landing collections, 2447 specimens (89.86%) of Anopheles minimus were obtained, followed by 119 Anopheles peditaeniatus (4.37%), 62 Anopheles maculatus (2.28%), 17 Anopheles dirus (0.6%), 15 Anopheles aconitus (0.5%) and 6 Anopheles sawadwongporni (0.2%) respectively. Seven putative malaria vectors, including An. minimus, An. dirus, An. baimaii, An. sawadwongporni, An. maculatus, An. pseudowillmori and An. aconitus were documented from this study and trophic behavior of each respective species were observed. Such information is definitely crucial for defining the vector capacity of each single species and for designing appropriate vector prevention and control strategies against target vector species.

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.

Behavioral responses of Anopheles species (Culicidae: Diptera) with varying surface exposure to pyrethroid-treated netting in an excito-repellency test system.

Indoor Residual Spray (IRS) with insecticides has been a procedure used for decades to protect humans from biting mosquitoes and potential vectors of pathogens. The objective of this study was to determine the responses of three wild-caught species of malaria vectors exposed to pyrethroids of three different surface coverage percents using an excito-repellency test box. Each species was exposed to three insecticide-treated surfaces at varying exposure levels (full coverage, 50%, and 25% of the maximum allowable by the test system) to a single standard field dose of either lambda-cyhalothrin or alpha-cypermethrin. Larger numbers of mosquitoes escaped the treated chambers in the direct contact test compared to the spatial repellent chambers in all three different treated surface exposures. No significant differences in the percent of escaped mosquitoes were detected in the 50% and full coverage surface coverage exposures, whereas the 25% coverage produced significantly lower avoidance responses for both compounds. This study found that varying levels of surface exposure with synthetic pyrethroids can impact the behavioral avoidance responses of Anopheles; however, it may also be possible to reduce the amount of coverage to achieve similar avoidance actions. This information may assist policy makers in designing more cost effective strategies involving residual insecticides to control mosquito vectors.

Comparative Excito-Repellency of Three Cambodian Plant-Derived Extracts Against Two Mosquito Vector Species, Aedes aegypti and Anopheles minimus.

A study of the behavioral responses of Aedes aegypti and Anopheles minimus to 3 Cambodian plant extracts at 3 different concentrations (1%, 2.5%, and 5%) was performed using an excito-repellency test system. These 3 plants were Strophanthus scandens, Capparis micracantha, and Dioscorea hispida, selected according to traditional healer's knowledge, bibliographic studies and market surveys. Results showed that S. scandens leaves' hexane extract was the only one to exert repellency against Ae. aegypti with 23.3% of escaped mosquitoes at a concentration of 5%. Capparis micracantha was responsible for an irritant activity against An. minimus with 20.2% of escaped mosquitoes at a concentration of 2.5% and 22.8% escaping at a concentration of 5%. Dioscorea hispida showed an irritant activity on both mosquito species with 23.2% of escaped Ae. aegypti at a concentration of 5% and about 20% of escaped An. minimus at 2.5% and 5%. This is the first report on the irritant and repellent activities of S. scandens , D. hispida , and C. micracantha against mosquito species.

Avoidance Behavior to Essential Oils by Anopheles minimus, a Malaria Vector in Thailand.

Essential oils extracted from 4 different plant species--citronella (Cymbopogon nardus), hairy basil (Ocimum americanum), sweet basil (Ocimum basilicum), and vetiver (Vetiveria zizanioides)-were investigated for their irritant and repellent activities against Anopheles minimus, using an excito-repellency test system. Pure essential oils were used in absolute ethanol at the concentrations of 0.5%, 1%, 2.5%, and 5% (v/v) compared with deet. At the lowest concentration of 0.5%, hairy basil displayed the best irritant and repellent effects against An. minimus. Citronella and vetiver at 1-5% showed strong irritant effects with>80% escape, while repellent effects of both oils were observed at 1% and 2.5% citronella (73-89% escape) and at 5% vetiver (83.9% escape). Sweet basil had only moderate irritant action at 5% concentration (69.6% escape) and slightly repellent on test mosquitoes (<50% escape). The results found that hairy basil, citronella, and vetiver are promising potential mosquito repellent products for protection against An. minimus.