Integrating 3D video tracking with the standard WHO tunnel assay — a proof-of-concept to support improving insecticide-treated nets for mosquito control

The World Health Organization (WHO) tunnel test is a standardised laboratory method for characterising the biological availability and potency of active ingredients on the surface of an insecticide-treated bed net (ITN) against host-seeking mosquitoes. However, the assay provides only endpoint measurements – the proportions of mosquitoes that are killed and blood-fed – and therefore offers no insight into how mosquitoes interact with the ITN. Therefore, complementary behavioural data could reveal, for example, the extent to which mosquitoes engage with the net – explaining differences in endpoint outcomes – or indicate the minimum duration required for the assay, thereby improving throughput. For capturing mosquito behaviour in detail, automated three-dimensional (3D) video tracking offers a powerful approach. Here, we present a proof-of-concept study combining the standard WHO tunnel assay with Trackit3D, a versatile tracking system, in a laboratory in Tanzania where tunnel assays are routinely conducted. The system tracked multiple mosquitoes simultaneously as they were attracted to a rabbit, despite typical fluctuations in power and lighting. The ability to obtain high-resolution trajectories within the WHO tunnel assay provides new opportunities to enhance the behavioural evaluation of vector control tools and to strengthen the interpretability and utility of the WHO tunnel assay.