Early-Evening Biting by Anopheles stephensi in Southern Ethiopia: A Challenge for Bed Net Use as Vector Control in Africa.

Anopheles stephensi is rapidly expanding across Africa, posing new challenges for malaria control. Its biting time patterns, however, remain poorly characterized, raising uncertainty about the effectiveness of bed nets against this invasive vector. To address this gap, we investigated diel biting activity, feeding propensity, and flight behavior using complementary behavioral assays on females reared from wild-caught larvae in Hawassa City, Ethiopia. Flight monitoring assays revealed that An. stephensi exhibited pronounced activity at dusk, beginning between 18:00 and 19:00 with scotophase onset, and little to no activity during photophase. Blood-feeding propensity, defined as the proportion of mosquitoes taking a blood meal when offered, peaked during early scotophase (18:00-22:00) at 33.3-51.7% but was markedly reduced during daylight hours (0-16.7%). Human landing catches in large-cage enclosures confirmed this early-evening activity: 83.5% of total landings occurred between 18:00 and 22:00, with a sharp peak at 18:00-19:00, corresponding to a mean biting rate of 17.8 bites per person per hour. These findings demonstrate that invasive An. stephensi primarily seek hosts and bite during the early evening, when people are often unprotected by bed nets. This behavior reduces the protective impact of conventional net-based interventions and underscores the need for African National Malaria Control Programs to deploy complementary measures such as spatial repellents and larval source management to mitigate early-evening transmission. Moreover, this study highlights the utility of integrated behavioral assays for estimating biting time, offering approaches that can be extended to other vector species across Africa.