Discovery of and characterization of genes related to insecticide resistance in Bactrocera dorsalis by functional genomic analysis of a de novo assembled transcriptome

Insecticide resistance has recently become a critical concern for the control of many insect pest species. Genome sequencing and global quantitation of gene expression through analysis of the transcriptome can supply useful information relevant to this challenging problem. The oriental fruit fly, Bactrocera dorsalis, is one of the world’s most destructive agricultural pests, and in recent years it has been widely used for studying genetic mechanisms of insecticide resistance. However, much of the molecular data available for this species is limited to genes identified through homology studies. To provide a broader pool of gene sequences of potential interest, we designed the current study to be the first report of a whole transcriptome analysis of B. dorsalis using de novo assemblage of short reads generated by next-generation sequencing (NGS). The transcriptome of B. dorsalis was initially constructed using Illumina’s Solexa sequencing technology. Qualified reads were assembled into contigs and into potential splicing variants (isotigs). A total of 29,067 of the isotigs have putative homologues in the nr database, and 11,073 of these correspond to distinct D. melanogaster proteins in the RefSeq database. Approximately 1,482 isotigs were shown to be complete (>0.8x) and are considered to represent B. dorsalis genes. Reducing the requirement for completeness to only >0.5x resulted in the identification of more than 6,000 sequences representing potential genes. We also observed a strong correlation between the completeness of the assembled sequences and the expression intensity of the transcripts. The assembled sequences were also used to identify large numbers of genes potentially belonging to families related to insecticide resistance. A total of 96 P450-, 42 GST- and 39 COE-related genes, representing three major enzyme families involved in insecticide metabolism and resistance, were identified. In addition, 37 isotigs were discovered to contain target site sequences related to four classes of insecticide resistance genes. Identified sequence motifs were analyzed to characterize the putative polypeptide translational products and associate them with specific genes and protein functions.