Detection and quantification of Anopheles gambiae sensu lato mosquito larvae in experimental aquatic habitats using environmental DNA (eDNA).

Abstract

<b>Background:</b> Growing insecticide resistance and changes in biting and resting behavior of malaria vectors threaten efficacy of insecticide treated nets and indoor residual spraying. Larval source management (LSM) is a promising approach that can target mosquitoes irrespective of their behavior as adults. However, the use of traditional monitoring methods for immature stages of <i>Anopheles</i> mosquitoes is a major challenge to LSM due to the variability in their breeding habitats. We evaluate the use of an environmental DNA (eDNA) analysis technique in monitoring <i>Anopheles gambiae</i> sensu lato larvae in experimental aquatic habitats. <b>Methods:</b> eDNA was simultaneously sampled and extracted from different volumes of water, number of larvae, and occupation time. Larval presence was detected using PCR and eDNA concentration in samples from 1 L habitats quantified using an <i>IGS</i> and <i>cyt b</i> TaqMan assays. The limit of detection of the two assays was tested and larval density correlated with eDNA positivity. <b>Results:</b> 74% of replicates in the 50 mL habitats were PCR positive with at least 6h required to get a signal from a single larva (0.02 larvae/mL). All 12 replicates where 1 L of water was used were positive with stronger PCR bands than replicates with the same larval density in 50 mL for 24 h. There was a correlation between larval densities and eDNA detection in both assays: <i>IGS</i>, <i>r</i> = 0.503, p = 0.047; and <i>cyt b,</i><i>r</i> = 0.558, p = 0.025. There was stochasticity in eDNA detection rates, using both PCR and qPCR across all the dilutions. <b>Conclusion:</b> This study has demonstrated the potential use of eDNA analysis for detection and quantification of <i>An. gambiae</i> s.s. mosquito larvae in aquatic habitats. The stochasticity observed in eDNA detection suggest that this technique is best for monitoring aquatic habitats with many larvae at low densities.