title: Transcriptional cellular responses in midgut tissue of Aedes aegypti larvae following intoxication with Cry11Aa toxin from Bacillus thuringiensis.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Departamento de Microbiología, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. postal 510-3, Cuernavaca, 62250, Morelos, Mexico.
authors.forename: Pablo Emiliano
authors.initials: PE
authors.lastname: Canton
authors.affiliation: Facultad de Ciencias, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, Distrito Federal, 04510, Mexico.
authors.forename: Angeles
authors.initials: A
authors.lastname: Cancino-Rodezno
authors.affiliation: Cell Biology and Neuroscience Department, University of California, Riverside, Riverside, CA, 92521, USA.
authors.forename: Sarjeet S
authors.initials: SS
authors.lastname: Gill
authors.affiliation: Departamento de Microbiología, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. postal 510-3, Cuernavaca, 62250, Morelos, Mexico.
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
authors.affiliation: Departamento de Microbiología, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. postal 510-3, Cuernavaca, 62250, Morelos, Mexico. bravo@ibt.unam.mx.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
mesh_terms: D000330:Aedes / Q000187:drug effects* / Q000235:genetics*; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000494:pharmacology*; D016000:Cluster Analysis; D019295:Computational Biology / Q000379:methods; D004351:Drug Resistance; D004731:Endotoxins / Q000494:pharmacology*; D041981:Gastrointestinal Tract / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D006460:Hemolysin Proteins / Q000494:pharmacology*; D059014:High-Throughput Nucleotide Sequencing; D020128:Inhibitory Concentration 50; D007306:Insecticides / Q000494:pharmacology*; D007814:Larva / Q000235:genetics*; D058977:Molecular Sequence Annotation; D015203:Reproducibility of Results; D059467:Transcriptome
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis