Integrating 3D video tracking with the standard WHO tunnel assay: a proof-of-concept to support improving...

The World Health Organization (WHO) tunnel test is a standardised laboratory assay used to characterise 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 killed and blood-fed - and therefore offers no insight into how mosquitoes interact with the ITN. Therefore, complementary behavioural data would be highly valuable, for example to reveal the extent to which mosquitoes engage with the net, thereby helping to explain differences in endpoint outcomes, or to determine the minimum assay duration required and thus improve 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 WHO tunnel assay with Trackit3D, a versatile tracking system, in a laboratory in Tanzania where tunnel assays are routinely performed. The system successfully tracked multiple mosquitoes simultaneously as they were attracted to a rabbit, including measuring the duration of contact with the net, despite typical fluctuations in power supply and lighting conditions. The ability to obtain high-resolution trajectories within the WHO tunnel assay provides new opportunities to enhance the behavioural evaluation of ITNs and strengthens both the interpretability and utility of the assay.