Comparison of different spectral ranges of UV-LED lighting for outdoor mosquito trapping in forested area in Thailand.

Mosquito surveillance is critical for actively tracking the location and monitoring population levels and the threat of mosquito-borne disease. Although light-emitting diodes (LEDs) light traps have grown in popularity, there is still a limited understanding of the application of light wavelengths for trapping nocturnally active wild mosquitoes in forest ecotypes. This study evaluated the performance of different UV wavelengths in trapping mosquito populations in a forested mountainous area in Nakhon Ratchasima province, Thailand. Traps with different UV wavelengths were deployed in 6 locations, following a 6 × 6 Latin square replicated 6 times over a total of 36 nights. Light traps were operated between 18:00 and 06:00 h from October 2022 to August 2023. Mosquitoes were separately collected from individual traps every 4 h at 22.00, 2.00, and 6.00 h. Mosquitoes were killed by placing in a freezer (- 20 °C) for at least 30 min and then were morphologically identified using illustrated keys for adult females. Traps fitted with the LED 365 wavelength light source were the most effective in capturing 790 (23.66%) of the total mosquitoes collected, followed by the UV fluorescent 632 (18.93%), with the other 4 LED wavelengths collecting between 16.89% (LED 385) and 12.64% (LED 375) of the mosquitoes. Culex was the most common genus, representing 56.00% of total mosquito abundance. LED 365 and LED 385 were comparable to the UV fluorescent traps (the standard reference). Optimal trapping times were during 18:00-22:00 h. Compared to the other wavelengths, LED 365 was significantly more effective at capturing Coquillettidia and Culex mosquitoes than the UV-based traps.

Dirus complex species identification PCR (DiCSIP) improves the identification of Anopheles dirus complex from the Greater Mekong Subregion.

BACKGROUND: The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deploying effective vector control measures. However, the original molecular identification assay, Dirus allele-specific polymerase chain reaction (AS-PCR), targeting the ITS2 region, has pronounced nonspecific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers to accurately identify species within the Anopheles dirus complex. METHODS: The AS-PCR reaction and thermal cycling conditions were modified to improve specificity for An. dirus member species identification. In silico analyses with Benchling and Primer-BLAST were conducted to identify problematic primers and design a new set for Dirus complex species identification PCR (DiCSIP). DiCSIP was then validated with laboratory and field samples of the An. dirus complex. RESULTS: Despite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications for Anopheles dirus, Anopheles scanloni, and Anopheles nemophilous. Subsequently, in silico analyses pinpointed problematic primers with high Guanine-Cytosine (GC) content and multiple off-target binding sites. Through a series of in silico analyses and laboratory validation, a new set of primers for Dirus complex species identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity to identify five complex member species in the GMS accurately. Validation with laboratory and field An. dirus complex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentified An. dirus as other species when used with different thermocyclers. CONCLUSIONS: The DiCSIP assay offers a significant improvement in An. dirus complex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission and prevent malaria re-introducing in the GMS.