Application of Environmental DNA (eDNA) in the Detection and Quantification of Anopheles Mosquito Larvae in Artificial Aquatic Habitats

Background Environmental DNA (eDNA) analysis is an emerging tool in ecological monitoring of aquatic habitats. When applied correctly, it can overcome the limitations of traditional aquatic sampling methods. This current study develops and validates a protocol for using eDNA analysis to detect and quantify mosquito larvae in laboratory and controlled field setting. Methods Species-specific PCR primers were designed from An. gambiae s.l and An. funestus, and qPCR primers for An. gambiae s.l using mitochondrial cyt b gene and validated on 1:10 and 1:100 dilutions of gDNA from both species. In the laboratory eDNA testing, An. gambiae s.s mosquito larvae (1 larva, 3 larvae and 6 larvae densities) were held in 45 mL of distilled water. One tube from each of the three densities was then sampled simultaneously at intervals of 1 h, 3 h, 6 h and 24 h.We also set up six 1 L habitats by adding 2, 5, 10 and 20 larvae into each with 1 negative control habitat and held for 24 h. eDNA from the lab habitats were concentration using two methods - filtration and centrifugation - and also extracted using either ChargeSwitch® or Nexttec and analyzed the eDNA using IGS assay and cyt b TaqMan assay.Field testing was done in 20 artificial ponds with varying total larval numbers and relative proportions of An. gambiae s.s. and An. arabiensis larvae of 6, 24, 48 and 96 larvae. Water samples (150 mL) were taken from the ponds daily over an 8day period. eDNA was extracted from them using the ChargeSwitch® and analyzed in a cyt b and IGS TaqMan assay. Results First test in mosquitoes was successful under laboratory conditions but inconclusive in the field trials. Both IGS and cyt b assays detected and quantified DNA at very low concentrations (0.156 pg). Contamination was a major issue in the field samples and affected any conclusion from the field samples. Conclusion We successfully showed that eDNA analysis can be used to detect and quantify mosquito species. More work in the protocol optimization need to be done to ensure smooth transition into semi field and field settings.