An innovative insecticidal approach based on plant protease inhibitor and Bt protoxins inhibits trypsin-like activity in zebrafish

The Leucaena leucocephala trypsin inhibitor (LTI) + Bacillus thuringiensis (Bt) protoxins mix has been proposed as a novel larvicide agent in order to control the vector mosquito of dengue virus, Aedes aegypti, in their aquatic breeding sites. However, use of this insecticide formulation has raised concerns about its impacts on aquatic biota. In this context, this work aimed to assess the effects of LTI and Bt protoxins, separately or in combination, in zebrafish, in regard to the evaluation of toxicity at early life stages and to the presence of LTI inhibitory effects on intestinal proteases of this fish. Results showed that LTI and Bt concentrations (250 mg/L, and 0.13 mg/L, respectively), and LTI + Bt mix (250 mg/L + 0.13 mg/L) -10 times superior to those with insecticidal action - did not cause death nor did it induce morphological changes during embryonic and larval development (3 to 144 h post-fertilization) of zebrafish. Molecular docking analyses highlighted a possible interaction between LTI and zebrafish trypsin, especially through hydrophobic interactions. In concentrations near to those with larvicidal action, LTI (0.1 mg/mL) was able to inhibit in vitro intestinal extracts of trypsin in female and male fish by 83 % and 85 %, respectively, while LTI + Bt mix promoted trypsin inhibition of 69 % in female and 65 % in male ones. These data show that the larvicidal mix can potentially promote deleterious effects to nutrition and survival in non-target aquatic organisms, especially those with trypsin-like dependent protein digestion.

Automated summary

This study aimed to assess the effects of Leucaena leucocephala trypsin inhibitor (LTI) and Bacillus thuringiensis (Bt) protoxins on zebrafish, specifically in terms of early life stage toxicity and LTI inhibitory effects on intestinal proteases. The results showed that LTI and Bt concentrations did not cause death or morphological changes in zebrafish. Molecular docking analysis suggested an interaction between LTI and zebrafish trypsin. The LTI + Bt mixture inhibited zebrafish trypsin activity. These findings indicate that the larvicidal mixture may have detrimental effects on non-target aquatic organisms, particularly those with trypsin-like dependent protein digestion.