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Cloning, expression and activity of ATP-binding protein in Bacillus thuringiensis toxicity modulation against Aedes aegypti.

ABSTRACT: BACKGROUND:Bacillus thuringiensis israelensis (Bti) is a widely used mosquitocidal microbial pesticide due to its high toxicity. ATP-binding proteins (ABP) are prevalently detected in insects and are related to reaction against Bti toxins. However, the function of ABP in mosquito biocontrol is little known, especially in Aedes aegypti. Therefore, this study aimed to clarify the function of ABP in Ae. aegypti against Bti toxin. RESULTS:Aedes aegypti ABP (GenBank: XM_001661856.2) was cloned, expressed and purified in this study. Far-western blotting and ELISA were also carried out to confirm the interaction between ABP and Cry11Aa. A bioassay of Cry11Aa was performed both in the presence and absence of ABP, which showed that the mortality of Ae. aegypti is increased with an increase in ABP. CONCLUSIONS:Our results suggest that ABP in Ae. aegypti can modulate the toxicity of Cry11Aa toxin to mosquitoes by binding to Bti toxin. This could not only enrich the mechanism of Bt toxin, but also provide more data for the biocontrol of this transmission vector.

SUBMITTER: Zhao GH

PROVIDER: S-EPMC6593554 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Cloning, expression and activity of ATP-binding protein in Bacillus thuringiensis toxicity modulation against Aedes aegypti.

Zhao Guo-Hui GH Liu Jian-Nan JN Hu Xiao-Hua XH Batool Khadija K Jin Liang L Wu Chen-Xu CX Wu Juan J Chen Hong H Jiang Xiao-Yan XY Yang Zhao-Hui ZH Huang Xian-Hui XH Huang En-Jiong EJ Yu Xiao-Qiang XQ Guan Xiong X Zhang Ling-Ling LL

Parasites & vectors 20190625 1

<h4>Background</h4>Bacillus thuringiensis israelensis (Bti) is a widely used mosquitocidal microbial pesticide due to its high toxicity. ATP-binding proteins (ABP) are prevalently detected in insects and are related to reaction against Bti toxins. However, the function of ABP in mosquito biocontrol is little known, especially in Aedes aegypti. Therefore, this study aimed to clarify the function of ABP in Ae. aegypti against Bti toxin.<h4>Results</h4>Aedes aegypti ABP (GenBank: XM_001661856.2) wa ...[more]

PMID: 31238963

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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.

Project description:Although much is known about the mechanism of action of Bacillus thuringiensis Cry toxins, the target tissue cellular responses to toxin activity is less understood. Previous transcriptomic studies indicated that significant changes in gene expression occurred during intoxication. However, most of these studies were done in organisms without a sequenced and annotated reference genome. A reference genome and transcriptome is available for the mosquito Aedes aegypti, and its importance as a disease vector has positioned its biological control as a primary health concern. Through RNA sequencing we sought to determine the transcriptional changes observed during intoxication by Cry11Aa in A. aegypti and to analyze possible defense and recovery mechanisms engaged after toxin ingestion.In this work the changes in the transcriptome of 4(th) instar A. aegypti larvae exposed to Cry11Aa toxin for 0, 3, 6, 9, and 12 h were analyzed. A total of 1060 differentially expressed genes after toxin ingestion were identified with two bioconductoR packages: DESeq2 and EdgeR. The most important transcriptional changes were observed after 9 or 12 h of toxin exposure. GO enrichment analysis of molecular function and biological process were performed as well as Interpro protein functional domains and pBLAST analyses. Up regulated processes include vesicular trafficking, small GTPase signaling, MAPK pathways, and lipid metabolism. In contrast, down regulated functions are related to transmembrane transport, detoxification mechanisms, cell proliferation and metabolism enzymes. Validation with RT-qPCR showed large agreement with Cry11Aa intoxication since these changes were not observed with untreated larvae or larvae treated with non-toxic Cry11Aa mutants, indicating that a fully functional pore forming Cry toxin is required for the observed transcriptional responses.This study presents the first transcriptome of Cry intoxication response in a fully sequenced insect, and reveals possible conserved cellular processes that enable larvae to contend with Cry intoxication in the disease vector A. aegypti. We found some similarities of the mosquito responses to Cry11Aa toxin with previously observed responses to other Cry toxins in different insect orders and in nematodes suggesting a conserved response to pore forming toxins. Surprisingly some of these responses also correlate with transcriptional changes observed in Bti-resistant and Cry11Aa-resistant mosquito larvae.

Dataset Information

Cloning, expression and activity of ATP-binding protein in Bacillus thuringiensis toxicity modulation against Aedes aegypti.

Publications

Cloning, expression and activity of ATP-binding protein in Bacillus thuringiensis toxicity modulation against Aedes aegypti.

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