Efficacy of a eugenol-based nanoemulsion formulation incorporating a mosquitocidal Lysinibacillus sp. strain VCRC B654 against Culex quinquefasciatus under laboratory and simulated field conditions.

Culex (Cx.) quinquefasciatus, an important vector of lymphatic filariasis, poses a major public health challenge due to the emergence of insecticide resistance and increasing environmental concerns associated with conventional control measures. This study aimed to characterize a soil isolate of Lysinibacillus sp. and evaluate its potential as a nanoformulated microbial biopesticide against Cx. quinquefasciatus. The isolate was characterized based on morphology, haemolytic activity, scanning electron microscopy, growth kinetics, protein profiling, and 16S rRNA gene sequencing. Lysinibacillus sp. VCRC B654 exhibited high larvicidal activity against late third-instar Cx. quinquefasciatus larvae (LC₅₀ = 0.0334 mg/L; LC₉₀ = 0.0783 mg/L), with comparatively lower activity against Anopheles stephensi and Aedes aegypti. A eugenol-based nanoemulsion containing bacterial biomass was prepared by ultrasonication and characterized for its physicochemical properties. The optimized formulation, comprising 0.5% (w/v) bacterial biomass, 5% eugenol, 15% Tween® 80, and 79.5% water, produced submicron droplets with a mean diameter of ≈460 nm, a polydispersity index of 0.418, and a zeta potential of - 8.9 mV. FTIR analysis confirmed the characteristic functional groups, while cryo-TEM demonstrated the incorporation of bacterial spores and crystal inclusions within spherical droplets. Compared with the unformulated bacterial biomass, the nanoemulsion enhanced larvicidal efficacy by 3.7-fold, with a relative median potency (RMP) of 3.814 (95% CI: 3.096-4.847), reducing the LC₅₀ and LC₉₀ values to 0.009 mg/L and 0.016 mg/L, respectively. The formulation retained more than 80% larvicidal activity for five weeks when applied at 8-fold the LC₉₀ under simulated field conditions, maintained spore viability for 12 months, and no mortality was observed among the tested non-target organisms during 72 h exposure. These findings demonstrate the potential of the eugenol-based nanoemulsion as a bacterial formulation for improved mosquito larval control and support further evaluation under diverse field conditions.