Persistent susceptibility of Aedes aegypti to eugenol

Abstract

Botanical insecticides are preferred for their environment and user-friendly nature. Eugenol is a plant-based monoterpene having multifarious biocidal activities. To understand whether eugenol would persistently work against Aedes aegypti, we performed larvicidal bioassays on thirty successive generations and determined median lethal concentration (LC50) on each generation. Results showed no apparent differences between LC50 at F0 (63.48 ppm) and F30 (64.50 ppm) indicating no alteration of susceptibility toward eugenol. To analyze, if eugenol has any effect on metabolic detoxification-associated enzymes, we measured esterases (alpha and beta), cytochrome P450, and GST activities from the survived larvae exposed to LC50 concentration from F0-F30. Results revealed a decrease of esterases, GST, and cytochrome P450 activities at the initial 4-8 generations and then a gradual increase as the generations progressed. GST activity remained significantly below the control groups. Synergists (TPP, DEM, and PBO) were applied along with eugenol at F30 and LC50 concentration, and the said enzyme activities were recorded. Results showed a noticeable decrease in LC50 and enzyme activities indicating effective inhibitions of the respective enzymes. Overall, present results inferred that eugenol would effectively work as a larvicide for a longer period in successive generations without initiating rapid resistance and therefore could be advocated for controlling A. aegypti.

Figure 1

Response of 4th instar larvae to a graded concentrations of eugenol over a generational time (± SE).

Figure 2

Bar diagram showing the median lethal concentration of eugenol on every fifth successive generations of A. aegypti upto F30 generations. 95% confidence intervals for each LC50 are presented in the form of error bars.

Figure 3

(A) α-esterase enzyme activity (± SE), (B) β-esterase enzyme activity (± SE) in continuously exposed (F0 to F30) A. aegypti larvae. The asterisks represents the significant difference between the experimental groups. Tukey’s post hoc test was employed to determine the significance in differences. The enzyme activity is expressed at the unit of μM of product formed/min/mg protein. *The mean difference is significant at the 0.05 level.

Figure 4

GST enzyme activity (± SE) in continuously exposed (F0 to F30) A. aegypti larvae. The asterisks represents the significant difference between the experimental groups. Tukey’s post hoc test was employed to determine the significance in differences. The enzyme activity is expressed at the unit of mM of conjugate produced/min/mg protein. *The mean difference is significant at the 0.05 level.

Figure 5

Cyt P450 enzyme activity (± SE) in continuously exposed (F0 to F30) A. aegypti larvae. The asterisks represents the significant difference between the experimental groups. Tukey’s post hoc test was employed to determine the significance in differences. The enzyme activity is expressed at the unit of cytochrome P450/min/mg protein. *The mean difference is significant at the 0.05 level.

Figure 6

Effect of synergists + eugenol on the median lethal concentration (LC50) on the F30 larvae of A. aegypti. As the 95CIs overlap between all three synergized eugenols, we have compared eugenol + PBO and Eugenol alone. 95% confidence intervals for each LC50 are presented in the form of error bars for each synergized treatment.

Figure 7

Effect of synergists (PBO, TPP and DEM) on the metabolic detoxification enzymes- esterases, GST and cytochrome P450 of A. aegypti. Asterisks represents the significance in difference and both directional vertical bar at the tip of each enzyme bar represents the error bars. Enzyme activity of esterases, cytochrome p450, and GST are expressed at the unit of μM of product formed/min/mg protein, cytochrome P450/min/mg protein, and mM of conjugate produced/min/mg protein, respectively.