eDNA-qPCR outperforms larval monitoring for detecting Aedes albopictus under tropical field conditions: associations with larval density and sun exposure

Abstract Mosquito vector surveillance is a major public health priority in tropical regions. Environmental DNA, a promising approach for detecting mosquito larvae in their aquatic breeding sites, remains poorly documented under tropical conditions and must overcome the challenges of DNA degradation and PCR inhibition. This study evaluated a qPCR assay targeting Aedes albopictus eDNA in larval breeding sites, compared its performance with conventional larval surveillance, and identified factors influencing detection and signal intensity. We conducted field validation at 28 breeding sites on La Réunion island over five months, from November 2025 to March 2026, totalling 102 site visits. At each visit, we filtered 120 mL water samples at each breeding site (Sterivex, 0.45 µm) and extracted DNA using a Macherey-Nagel NucleoSpin eDNA Water Kit. We amplified DNA by qPCR using Environmental Master Mix in triplicate, alongside larval surveys. A site was considered eDNA-positive if at least two out of three replicates amplified beyond the detection threshold, established based on a calibration curve. We assessed predictors of detection and signal intensity using mixed models. In the field, eDNA-qPCR detected Ae. albopictus in 64.7% of site visits, significantly more than larval surveys (39.2%, McNemar’s test, X2(1) = 16.45, p < 0.001). Agreement between the two methods was fair (Cohen’s Kappa = 0.30, 95% CI [0.14–0.46]), reflecting their differing temporal sensitivity rather than assay unreliability. Positive and negative percentage agreement were 85% (95% CI: [70.9, 92.9] %) and 48.4% (95% CI: [36.4, 60.6] %), respectively. Larval density was negatively correlated with Ct values (rho=-0.46, p = 0.007), confirming a dose-dependent relationship between larval abundance and eDNA signal. Sun exposure affected eDNA signal intensity, but not detection probability. eDNA-qPCR significantly outperformed larval surveys for detecting Ae. albopictus under tropical field conditions, supporting its use for early detection of invasive vectors and monitoring of vector-control interventions.