The Two Tube Volatile Assay: a non-contact benchtop bioassay for monitoring susceptibility to transfluthrin
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Introduction
Progress against malaria has stalled since 2015, with insecticide resistance a key driver. Spatial emanators release volatile insecticides into the air, exposing mosquitoes through a route distinct from the tarsal contact used by treated nets, indoor residual spraying, and standard bioassays. Transfluthrin is currently monitored using the WHO bottle bioassay, which combines contact and vapour exposure and cannot isolate airborne effects. A scalable, vapour-only method is needed to characterise susceptibility to volatile pyrethroids.
Methods
We adapted the WHO tube bioassay to deliver non-contact transfluthrin vapour exposure without contacting treated papers. Non-blood-fed female Anopheles were exposed to acetone control or transfluthrin papers at 0.005, 0.1 and 2 mg/paper. Knockdown was recorded over 60 minutes and mortality at 24 hours. Testing spanned three laboratories (LSTM, AIRID and KEMRI) using susceptible reference and resistant lab colonies. Dose response models estimated EC□□ for knockdown (EC□□KD) and mortality (EC□□Mort), with resistance ratios (RR) against the local susceptible reference.
Results
The assay generated clear concentration- and time-dependent responses. At LSTM, Kisumu gave an EC□□KD of 0.052 and EC□□Mort of 0.054 mg/paper; Siaya was comparable (RR□□KD 1.17, RR□□Mort 0.89), whereas Tiassalé 13 and KDR showed reduced susceptibility (RR□□KD 2.42 and 2.85; RR□□Mort 5.56 and 6.65). At AIRID, the resistant Cové strain did not reach 50% knockdown or mortality. At KEMRI, Siaya showed reduced susceptibility (RR□□KD 3.36/RR□□Mort 5.65). Kisumu at AIRID and KEMRI was comparable to LSTM (EC□□KD 0.007/EC□□Mort 0.030; EC□□KD 0.044/EC□□Mort 0.023). Despite inter-laboratory variation, susceptible strains remained distinguishable from resistant ones. Lower RRs than for contact pyrethroids suggest contact-based phenotypes may not predict vapour susceptibility.
Conclusion
The assay provides a practical benchtop method using standard WHO hardware, distinguishing susceptible from resistant strains across three laboratories. Twenty-four-hour mortality is the recommended endpoint, 60-minute knockdown secondary. Not yet validated, it provides a foundation for further validation and adaptation to other actives.
