Towards environmental detection, quantification, and molecular characterization of Anopheles stephensi and Aedes aegypti from larval breeding sites

The invasion and establishment of An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, which require little prior knowledge of mosquito larval morphology and are quick and easy to implement at the sampling stage. This study validated the use of environmental DNA (eDNA) for simultaneous detection of An. stephensi and Ae. aegypti in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in one litre of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 32 second instar larvae in 50ml of water. An. stephensi eDNA, derived from emergent pupae for 24 hours, was remarkably stable, and still detectable almost two weeks later. eDNA surveillance has the potential to be implemented in local endemic communities as part of citizen science initiatives, and/or in cargo ports at points of country entry, to monitor the spread of invasive malaria vector species. Further studies are required to validate the feasibility of this technique under field conditions.