Novel Point Mutations of the ace-1 Gene of Aedes aegypti Larva Treated with Methanolic Extract of Citrus hystrix

Introduction: The mosquito species of Ae. aegypti is a vector of arthropod-borne diseases such as dengue haemorrhagic fever. Acetylcholinesterase (AChE) enzyme in Ae. aegypti that encoded by the ace-1 gene. Damage in the ace-1 gene as target of insecticide lead to the loss of the normal structure and function of AChE. However, damage in the ace-1 gene remains uncharacterised. The main aim of this study was to find out the point mutations of ace-1 gene in Ae. aegypti larvae treated with methanolic extract of Citrus hystrix leaves. Method: This experiment using a completely randomized design with two treatment groups. A container containing lethal concentration 50 of methanolic extract of C. hystrix leaves, and a control group containing only water with 0.5% Tween-20. Each group contained 50 third instar larvae of Ae. aegypti, and each group was repeated four times. Observation was performed for 24 h for the number of survived and dead larvae. Survived and dead larvae were collected prior to the DNA extraction, PCR, electrophoresis, and sequencing. The sequences of those two groups were then compared to determine the point mutations using genetyx ver 12. Results: The PCR products of both groups showed clear bands of 500-600 bp long. Furthermore, the presence of the mutation was confirmed by sequencing the PCR product of ace-1 between each treatment group. The survived larva in the extract-treated group showed more point mutation compared with that of dead larvae. Conclusions: This first report indicated that many mutations in the form of deletions and insertions in nitrogenous bases and different amino acid variations of the ace-1 gene of third instar larvae of Ae. aegypti after 24 h treated with methanolic extract of C. hystrix leaves than those in control group.