Abstract
BackgroundThe 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.ResultsDeep 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.ConclusionsHere, 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.
Highlights
The New World screw-worm (NWS), Cochliomyia hominivorax, is one of the most important myiasiscausing flies, causing severe losses to the livestock industry
Deep sequencing on the 454 platform of the three normalized libraries generated a total of 548,940 reads of raw nucleotide sequence data with an average read length of 184 bp after the removal of low score ends (Table 1)
The average contig size obtained is consistent with previous reports describing expressed sequence tags (ESTs) sequencing using the 454 technology for different insect species, Melitaea cinxia [24], Sarcophaga crassipalpis [22] and Chrysomela tremulae [21]
Summary
The New World screw-worm (NWS), Cochliomyia hominivorax, is one of the most important myiasiscausing flies, causing severe losses to the livestock industry. As new sequencing technologies became less expensive, it is possible to generate genomic information from non-model species. This holds a great promise for several species with medical, veterinary or economic importance. NWS myiasis is caused by the larval stage of the fly infesting tissues of warmblooded vertebrates Such infestations cause significant losses in the livestock industry through morbidity, mortality and the cost of treating infested animals. This insect pest represents a serious public health problem in the Caribbean region, where screw-worm infestations in humans are frequently reported [2]. In South America and in the Caribbean region, this pest continues to affect the development of the livestock sector and wider economic development
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