Abstract
BackgroundBacillus thuringiensis svar. israelensis (Bti) is an effective and safe biolarvicide to control Aedes aegypti. Its mode of action based on four protoxins disfavors resistance; however, control in endemic areas that display high mosquito infestation throughout the year requires continuous larvicide applications, which imposes a strong selection pressure. Therefore, this study aimed to investigate the effects of an intensive Bti exposure on an Ae. aegypti strain (RecBti), regarding its susceptibility to Bti and two of its protoxins tested individually, to other control agents temephos and diflubenzuron, and its profile of detoxifying enzymes.MethodsThe RecBti strain was established using a large egg sample (10,000) from Recife city (Brazil) and more than 290,000 larvae were subjected to Bti throughout 30 generations. Larvae susceptibility to larvicides and the activity of detoxifying enzymes were determined by bioassays and catalytic assays, respectively. The Rockefeller strain was the reference used for these evaluations.ResultsBti exposure yielded an average of 74% mortality at each generation. Larvae assessed in seven time points throughout the 30 generations were susceptible to Bti crystal (resistance ratio RR ≤ 2.8) and to its individual toxins Cry11Aa and Cry4Ba (RR ≤ 4.1). Early signs of altered susceptibility to Cry11Aa were detected in the last evaluations, suggesting that this toxin was a marker of the selection pressure imposed. RecBti larvae were also susceptible (RR ≤ 1.6) to the other control agents, temephos and diflubenzuron. The activity of the detoxifying enzymes α- and β-esterases, glutathione-S-transferases and mixed-function oxidases was classified as unaltered in larvae from two generations (F19 and F25), except for a β-esterases increase in F25.ConclusionsProlonged exposure of Ae. aegypti larvae to Bti did not evolve into resistance to the crystal, and no cross-resistance with temephos and diflubenzuron were recorded, which supports their sustainable use with Bti for integrated control practices. The unaltered activity of most detoxifying enzymes suggests that they might not play a major role in the metabolism of Bti toxins, therefore resistance by this mechanism is unlikely to occur. This study also highlights the need to establish suitable criteria to classify the status of larval susceptibility/resistance.
Highlights
Bacillus thuringiensis svar. israelensis (Bti) is an effective and safe biolarvicide to control Aedes aegypti
Data from our study demonstrated that Bti-selection was not correlated with a marked increase in the activity of detoxifying enzymes in Bti-exposed larvae and no alteration in the susceptibility to the insecticidal compounds tested was detected
The RecBti strain exposed to Bti crystals did not display a loss of susceptibility to Bti or to two of its protoxins after 30 generations of intensive selection pressure with a Bti-based larvicide
Summary
Bacillus thuringiensis svar. israelensis (Bti) is an effective and safe biolarvicide to control Aedes aegypti. One of the most remarkable features of Bti is the complex composition of the crystal, which contains protoxins belonging to the 3-domain Cry toxin family (Cry11Aa, Cry4Ba and Cry4Aa) and the cytolytic toxin (Cyt1Aa) [16, 17] These protoxins act in synergy, as the individual components exhibit lower toxicity than the whole set of components from the crystal, which displays the highest larvicidal action [18]. The major steps of Bti’s mode of action include the ingestion of the crystals by larvae, their solubilization in the midgut and the release of the protoxins that are proteolytically converted into toxins These activated toxins interact with midgut receptors, insert in the membranes provoking pore formation that leads to cell permeability and osmotic lyses that damage the epithelium [19]. The signaling pathway, proposes that the monomeric activated toxins bind to the cadherins receptors and activates an Mg2+
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