An evaluation of longitudinal Anopheles stephensi egg viability and resistance to desiccation over time

Abstract Anopheles stephensi , a malaria vector in South Asia and parts of the Middle East, has been detected as an invasive species in numerous African countries in recent years. It threatens to increase malaria disease burden and reverse gains made in malaria control and elimination over the past decades on the continent. To halt further expansion, it is critical to understand the biological characteristics that may have facilitated An. stephensi range expansion to Africa. In its invasive range, An. stephensi larvae have been found to colonize artificial containers, many of which are shared with Aedes aegypti . The success of Ae. aegypti as an invasive vector is often attributed to the use of artificial containers and the ability of Ae. aegypti eggs to remain viable in the absence of water for months. While An. stephensi is found in artificial containers, it is unclear whether the eggs can remain viable without water for extended periods. Anopheles stephensi eggs were reported to remain viable in soil for up to 12 days in a study done almost 100 years ago, but this work has not been revisited since. Thus, in this present study, we used egg batches (>100 eggs per batch) from two laboratory strains of An. stephensi (SDA500 and STE2) from South Asia and one Ae. aegypti strain (LVP-IB12) to evaluate 1) whether An. stephensi eggs can remain viable like Ae. aegypti when egg substrates are completely dried following standard insectary methods for drying out Aedes aegypti egg sheets, and 2) assess egg viability duration at varying temperatures (15°C, 20°C, 25°C 30°C, 35°C) when eggs are held on a moistened substrate in a high humidity environment. Anopheles stephensi egg viability and subsequent larval survival was observed consistently when moistened egg sheets were held at 15°C in a high humidity (>75% RH) environment for up to 14 days in both strains. Anopheles stephensi eggs were not viable when dried following standard insectary Aedes aegypti methods for drying out egg sheets, except when the protocol was amended to include a 15°C storage temperature. Though egg viability and larval survival was observed in the amended protocol for SDA500 and STE2 (16% and 21% respectively), it was significantly less than that of LVP-IB12 (83%) and was only observed in the egg batches stored for the shortest timepoint (seven days post egg collection, three days post complete drying of egg sheets). These findings suggest that An. stephensi may remain viable if eggs are transported under ideal conditions (15°C and >75% RH) through trade or commerce routes. Thus, the persistence of An. stephensi eggs in the absence of water should be considered in programs that engage in surveillance and control of An. stephensi in Africa.