title: Deep sequencing of New World screw-worm transcripts to discover genes involved in insecticide resistance.
abstract: BACKGROUND
  The New World screw-worm (NWS), Cochliomyia hominivorax, is one of the most important myiasis-causing flies, causing severe losses to the livestock industry. In its current geographical distribution, this species has been controlled by the application of insecticides, mainly organophosphate (OP) compounds, but a number of lineages have been identified that are resistant to such chemicals. Despite its economic importance, only limited genetic information is available for the NWS. Here, as a part of an effort to characterize the C. hominivorax genome and identify putative genes involved in insecticide resistance, we sampled its transcriptome by deep sequencing of polyadenylated transcripts using the 454 sequencing technology.
  
  
  RESULTS
  Deep sequencing on the 454 platform of three normalized libraries (larval, adult male and adult female) generated a total of 548,940 reads. Eighteen candidate genes coding for three metabolic detoxification enzyme families, cytochrome P450 monooxygenases, glutathione S-transferases and carboxyl/cholinesterases were selected and gene expression levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Of the investigated candidates, only one gene was expressed differently between control and resistant larvae with, at least, a 10-fold down-regulation in the resistant larvae. The presence of mutations in the acetylcholinesterase (target site) and carboxylesterase E3 genes was investigated and all of the resistant flies presented E3 mutations previously associated with insecticide resistance.
  
  
  CONCLUSIONS
  Here, we provided the largest database of NWS expressed sequence tags that is an important resource, not only for further studies on the molecular basis of the OP resistance in NWS fly, but also for functional and comparative studies among Calliphoridae flies. Among our candidates, only one gene was found differentially expressed in resistant individuals, and its role on insecticide resistance should be further investigated. Furthermore, the absence of mutations in the OP target site and the high frequency of mutant carboxylesterase E3 indicate that metabolic resistance mechanisms have evolved predominantly in this species.
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
authors.forename: Renato A
authors.initials: RA
authors.lastname: Carvalho
authors.forename: Ana Maria L
authors.initials: AM
authors.lastname: Azeredo-Espin
authors.forename: Tatiana T
authors.initials: TT
authors.lastname: Torres
mesh_terms: D000818:Animals; D001483:Base Sequence; D030541:Databases, Genetic; D004006:Dichlorvos / Q000633:toxicity; D004175:Diptera / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004331:Drosophila melanogaster / Q000187:drug effects / Q000235:genetics; D020224:Expressed Sequence Tags; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D015723:Gene Library; D017344:Genes, Insect / Q000235:genetics*; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D007305:Insecticide Resistance / Q000187:drug effects / Q000235:genetics*; D007814:Larva / Q000187:drug effects / Q000235:genetics; D008297:Male; D058977:Molecular Sequence Annotation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D012727:Sex Characteristics
chemical_list: D012333:RNA, Messenger; D004006:Dichlorvos