Dataset Information

Transcriptomic Analysis of Aedes aegypti in Response to Mosquitocidal Bacillus thuringiensis LLP29 Toxin.

ABSTRACT: Globally, Aedes aegypti is one of the most dangerous mosquitoes that plays a crucial role as a vector for human diseases, such as yellow fever, dengue, and chikungunya. To identify (1) transcriptomic basis of midgut (2) key genes that are involved in the toxicity process by a comparative transcriptomic analysis between the control and Bacillus thuringiensis (Bt) toxin (LLP29 proteins)-treated groups. Next-generation sequencing technology was used to sequence the midgut transcriptome of A. aegypti. A total of 17130 unigenes, including 574 new unigenes, were identified containing 16358 (95.49%) unigenes that were functionally annotated. According to differentially expressed gene (DEG) analysis, 557 DEGs were annotated, including 226 upregulated and 231 downregulated unigenes in the Bt toxin-treated group. A total of 442 DEGs were functionally annotated; among these, 33 were specific to multidrug resistance, 6 were immune-system-related (Lectin, Defensin, Lysozyme), 28 were related to putative proteases, 7 were lipase-related, 8 were related to phosphatases, and 30 were related to other transporters. In addition, the relative expression of 28 DEGs was further confirmed through quantitative real time polymerase chain reaction. The results provide a transcriptomic basis for the identification and functional authentication of DEGs in A. aegypti.

SUBMITTER: Batool K

PROVIDER: S-EPMC6107635 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Transcriptomic Analysis of Aedes aegypti in Response to Mosquitocidal Bacillus thuringiensis LLP29 Toxin.

Batool Khadija K Alam Intikhab I Wu Songqing S Liu Wencheng W Zhao Guohui G Chen Mingfeng M Wang Junxiang J Xu Jin J Huang Tianpei T Pan Xiaohong X Yu Xiaoqiang X Guan Xiong X Xu Lei L Zhang Lingling L

Scientific reports 20180823 1

Globally, Aedes aegypti is one of the most dangerous mosquitoes that plays a crucial role as a vector for human diseases, such as yellow fever, dengue, and chikungunya. To identify (1) transcriptomic basis of midgut (2) key genes that are involved in the toxicity process by a comparative transcriptomic analysis between the control and Bacillus thuringiensis (Bt) toxin (LLP29 proteins)-treated groups. Next-generation sequencing technology was used to sequence the midgut transcriptome of A. aegypt ...[more]

PMID: 30140020

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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.

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:The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered "net like" structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

Dataset Information

Transcriptomic Analysis of Aedes aegypti in Response to Mosquitocidal Bacillus thuringiensis LLP29 Toxin.

Publications

Transcriptomic Analysis of Aedes aegypti in Response to Mosquitocidal Bacillus thuringiensis LLP29 Toxin.

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