Dataset Information

Long-term exposure of Aedes aegypti to Bacillus thuringiensis svar. israelensis did not involve altered susceptibility to this microbial larvicide or to other control agents.

ABSTRACT:

Background

Bacillus 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.

Methods

The 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.

Results

Bti 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.

Conclusions

Prolonged 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.

SUBMITTER: Carvalho KDS

PROVIDER: S-EPMC6311009 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Insect microbiota is a dynamic microbial community that can actively participate in defense against pathogens. Bacillus thuringiensis (Bt) is a natural entomopathogen widely used as a bioinsecticide for pest control. Although Bt's mode of action has been extensively studied, whether the presence of microbiota is mandatory for Bt to effectively kill the insect is still under debate. An association between a higher tolerance and a modified microbiota was already evidenced but a critical point remained to be solved: is the modified microbiota a cause or a consequence of a higher tolerance to Bt? METHODS:In this study we focused on the mosquito species Aedes aegypti, as no work has been performed on Diptera on this topic to date, and on B. thuringiensis israelensis (Bti), which is used worldwide for mosquito control. To avoid using antibiotics to cure bacterial microbiota, mosquito larvae were exposed to an hourly increasing dose of Bti during 25 hours to separate the most susceptible larvae dying quickly from more tolerant individuals, with longer survival. RESULTS:Denaturing gradient gel electrophoresis (DGGE) fingerprinting revealed that mosquito larval bacterial microbiota was strongly affected by Bti infection after only a few hours of exposure. Bacterial microbiota from the most tolerant larvae showed the lowest diversity but the highest inter-individual differences. The proportion of Bti in the host tissue was reduced in the most tolerant larvae as compared to the most susceptible ones, suggesting an active control of Bti infection by the host. CONCLUSIONS:Here we show that a modified microbiota is associated with a higher tolerance of mosquitoes to Bti, but that it is rather a consequence of Bti infection than the cause of the higher tolerance. This study paves the way to future investigations aiming at unraveling the role of host immunity, inter-species bacterial competition and kinetics of host colonization by Bti that could be at the basis of the phenotype observed in this study.

Project description:BackgroundAedes aegypti is the vector of dengue virus, and its control is essential to prevent disease transmission. Among the agents available to control this species, biolarvicides based on Bacillus thuringiensis serovar israelensis (Bti) are an effective alternative to replace the organophosphate temephos for controlling populations that display resistance to this insecticide. The major goal of this study was to determine the baseline susceptibility of Brazilian Ae. aegypti populations to Bti, taking into account their background in terms of larvicide exposure, status of temephos resistance and the level of activity of detoxifying enzymes involved in metabolic resistance to insecticides.MethodsPopulation samples were established under insectarium conditions. Larval susceptibility to temephos and Bti was evaluated through bioassays and lethal concentrations of these compounds were determined. Biochemical assays were performed to determine the specific activity of five detoxifying enzymes in these samples.ResultsFourteen populations were characterized and, except for one case, all displayed resistance to temephos. Most populations were classified as highly resistant. The populations also showed increased activity of one or more detoxifying enzymes (glutathione-S-transferases, esterases and mixed function oxidases), regardless of their temephos resistance status. All populations analyzed were susceptible to Bti, and the lethal concentrations were similar to those detected in two laboratory susceptible colonies. The response to Bti showed little variation. A maximum resistance ratio of 2.1 was observed in two untreated populations, while in two Bti-treated populations, the maximum resistance ratio was 1.9. No positive correlation was found between temephos resistance, increased activity of detoxifying enzymes, and susceptibility to Bti.ConclusionsData from this study show that all populations were susceptible to Bti, including twelve untreated and two treated populations that had been exposed to this agent for more than ten years. The temephos resistance and increased activity of detoxifying enzymes observed in thirteen populations was not correlated with changes in susceptibility to Bti. Our data show a lack of cross-resistance between these two compounds; thus, Bti can be used in an integrated control program to fight Ae. aegypti and counteract the temephos resistance that was found among all populations analyzed.

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