Effects of a Proteinase Inhibitor from Inga laurina Seeds (ILTI) on Aedes aegypti Larval Development

Abstract

Aedes aegypti (Linnaeus, 1762) is Brazil's primary vector of epidemiologically significant arboviruses such as yellow fever, dengue, Zika, and chikungunya. Despite using conventional chemical control measures, this species has developed resistance to standard chemical insecticides, prompting the search for natural larvicidal compounds. Plant protease inhibitors offer an insecticidal alternative as the primary digestive enzymes in the midgut of Ae. aegypti are proteases (trypsin and chymotrypsin). Ae. aegypti larvae fed with ILTI, a Kunitz-type trypsin inhibitor derived from Inga laurina seeds, at concentrations between 0.03 mg of protein per mL (mgP/mL) and 0.12 mgP/mL, exhibited delayed larval development, with a lethal concentration (LC₅₀) of 0.095 mgP mL^-1 of ILTI for 50% of fourth-instar larvae (L₄). The ex vivo assay indicated that ILTI effectively inhibited the activity of larval trypsin, which remained susceptible to the inhibitor. Additionally, molecular modelling and docking studies were conducted to predict the three-dimensional ILTI/enzyme molecular complexes at the atomic level. Therefore, the results demonstrate that ILTI functions as a protease inhibitor in this species, presenting itself as a promising larvicidal tool in the control of Ae. aegypti.

Keywords: Aedes aegypti; Kunitz-type inhibitor; digestive enzyme; insecticide; molecular docking; protease.

Figure 1

Chronic effect of different concentrations of ILTI on the ongoing development of Ae. aegypti larvae exposed to distilled water (control) or ILTI (0.03, 0.06, 0.09 and 0.12 mgP mL⁻¹) after 48 h (A), 96 h (B) and 144 h (C). Different letters indicate a significant difference between larvae from the same treatment (ANOVA, p < 0.05 followed by Turkey’s post-test). L₁ (1st larval instar); L₂ (2nd larval instar); L₃ (3rd larval instar); L₄ (4th larval instar).

Figure 2

Biological effect of ILTI (0.03, 0.06, 0.09 and 0.12 mgP mL⁻¹) on Ae. aegypti larval weight after 48 h, 96 h and 144 h of treatment. Bars indicate mean ± SD. Different letters indicate a significant difference (p < 0.05) between larvae from the same treatment time (ANOVA, p < 0.05).

Figure 3

Biological effect of ILTI (0.03, 0.06, 0.09, and 0.12 mgP mL⁻¹) on larval survival (%) of Ae. aegypti L₄ after 48 h, 96 h, and 144 h of treatment. Bars indicate mean ± SD. Different letters indicate a significant difference (p < 0.05) between larvae from the same treatment time (ANOVA, p < 0.05).

Figure 4

The biological effect of ILTI (0.03, 0.06, and 0.09 mgP mL⁻¹) on the residual activity of trypsin and chymotrypsin of Ae. aegypti larvae, expressed in the amount of substrate BApNA (A) and SAApNA (B) hydrolysed per minute. Different letters indicate a significant difference (p < 0.05) between larvae of the same instar.

Figure 4

The biological effect of ILTI (0.03, 0.06, and 0.09 mgP mL⁻¹) on the residual activity of trypsin and chymotrypsin of Ae. aegypti larvae, expressed in the amount of substrate BApNA (A) and SAApNA (B) hydrolysed per minute. Different letters indicate a significant difference (p < 0.05) between larvae of the same instar.

Figure 5

Effect of ILTI on acetylcholinesterase activity in Ae. aegypti larvae L₃ and L₄, respectively, treated with distilled water (control) and ILTI (0.03, 0.06, 0.09 and 0.12 mgP mL⁻¹). Bars indicate residual enzymatic activity, expressed in nmol of dithionitrobenzoic acid released per minute. Equal letters indicate no significant difference (ANOVA, p < 0.05) when compared to controls.

Figure 6

Effect of ILTI on the acid and alkaline phosphatase enzymes of Ae. aegypti. The fourth instar larvae remained in contact with distilled water (control) or ILTI (0.03, 0.06, 0.09 and 0.12 mgP mL⁻¹) for 48 h. The results indicate the activity of phosphatases, measured in µmol of p-nitrophenol per µg of protein released per minute. Different letters indicate a significant difference (p < 0.05) between groups of the same enzyme class.

Figure 7

Ex vivo inhibition of trypsin activity in L₄ midguts from ILTI-fed larvae (0.06 mgP mL⁻¹ diet) by serine protease inhibitors (100 mM TLCK, 1 mM PMSF, or 1 mgP mL⁻¹ ILTI). Different letters indicate significant differences between treatments within each dietary group (ANOVA/ Tukey, p < 0.05).

Figure 8

Three-dimensional theoretical models for ILTI (A), Ae. aegypti trypsin (B) and Ae. aegypti chymotrypsin (C). The predicted conformations for ILTI/trypsin (D) and ILTI/ chymotrypsin (E) complexes are also represented.