Background The Barbirostris Complex comprises six formally described species that cannot be differentiated based on morphology alone. Out of these six species, two have been reported as putative malaria vectors, An. campestris and An. wejchoochotei . Five species are present in Thailand, An. barbirostris , An. campestris , An. dissidens , An. saeungae and An. wejchoochotei , while An . vanderwulpi occurs in Indonesia. As these species cannot be accurately differentiated by morphological characters, there is a crucial lack of information on their bionomics and role in the transmission of malaria and filariasis agents. Results For differentiating the six species, an allele-specific amplification (AS-PCR) based on the second internal transcribed spacer (ITS2) sequence was developed. From 862 mosquitoes in the Barbirostris Complex collected in 23 provinces throughout Thailand, the AS-PCR was able to identify five species and its validation was undertaken on 185 specimens. Conclusions This multiplex-PCR assay is potentially able to definitely identify all six species of the Barbirostris Complex and was validated on five species present 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.

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 (LC 50 and LC 75 ), lethal concentrations (LC 99 ) 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 LC 50 , 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 LC 50 and LC 99 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 LC 50 . 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.

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Excito-repellency activity of plant extracts have been increasingly studied as mosquito repellents. In this study, the crude extract of Andrographis paniculata was evaluated for its noncontact repellency, contact excitation (irritancy + repellency), and knockdown/toxicity response against five colonized mosquitoes; Aedes aegypti (L.), Aedes albopictus (Skuse), Anopheles dirus Peyton & Harrison, Anopheles epiroticus Linton & Harbach, and Culex quinquefasciatus Say (Diptera: Culicidae) using an excito-repellency assay system under laboratory-controlled conditions. The escape responses were observed at four different concentrations (0.5–5.0% w/v) with A. paniculata showing strong spatial repellency against Ae. albopictus (96.7% escape) and Ae. aegypti (71.7% escape) at the 2.5% and 0.5% concentrations, respectively. At 0.5% and 5.0% concentrations, the greatest repellency was seen for An. dirus (48.2% escape) and Cx. quinquefasciatus (59.7% escape), respectively. Comparatively, low repellency action was observed against An. epiroticus (1.6–15.0% escape). Escape in contact assays (before adjustment) was generally less pronounced compared to noncontact spatial repellency, with Ae. albopictus showing highest percent escape (71.4% escape) in the contact assay at 1.0% concentration. After adjusting for spatial repellency, escape due to contact irritancy alone was either not present or an insignificant contribution to the overall avoidance response for all species. No knockdown or mortality at 24-h postexposure was observed in any trials. These findings indicate that the A. paniculata crude extract is more active against day-biting mosquitoes; however, this may be a reflection of the time of testing. This study demonstrates compelling evidence that A. paniculata extract performs primarily as a spatial repellent. Further investigations exploring the use A. paniculata as a potential active ingredient in repellent products are needed.