Discovery of Genes Related to Insecticide Resistance in Bactrocera dorsalis by Functional Genomic Analysis of a De Novo Assembled Transcriptome

Abstract

Insecticide resistance has recently become a critical concern for control of many insect pest species. Genome sequencing and global quantization of gene expression through analysis of the transcriptome can provide useful information relevant to this challenging problem. The oriental fruit fly, Bactrocera dorsalis, is one of the world's most destructive agricultural pests, and recently it has been used as a target for studies of genetic mechanisms related to insecticide resistance. However, prior to this study, the molecular data available for this species was largely limited to genes identified through homology. To provide a broader pool of gene sequences of potential interest with regard to insecticide resistance, this study uses whole transcriptome analysis developed through de novo assembly 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 potential splicing variants (isotigs). A total of 29,067 isotigs have putative homologues in the non-redundant (nr) protein database from NCBI, and 11,073 of these correspond to distinct D. melanogaster proteins in the RefSeq database. Approximately 5,546 isotigs contain coding sequences that are at least 80% complete and appear to represent B. dorsalis genes. We 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 90 P450-, 42 GST-and 37 COE-related genes, representing three major enzyme families involved in insecticide metabolism and resistance, were identified. In addition, 36 isotigs were discovered to contain target site sequences related to four classes of resistance genes. Identified sequence motifs were also analyzed to characterize putative polypeptide translational products and associate them with specific genes and protein functions.

Analysis of Insecticide Resistance-Related Genes of the Carmine Spider Mite Tetranychus cinnabarinus Based on a De Novo Assembled Transcriptome

“…Sequence alignment was performed for 10 chosen full-length mRNA sequences and 23 corresponding annotated unigenes from the transcriptome assembly, in which 95% identity and 80% alignment length were set as thresholds. The average identity between the 10 previously identified CSM cDNA sequences in the NCBI nucleotide database and the 23 assembled sequences identified in the CSM transcriptome was 99.21% (Table 2), which is similar to the level of sequencing accuracy reported by other studies for Illumina technology [28], [29]. Sequencing error or nucleotide polymorphisms may be responsible for the nucleotide diversity between assembled sequences and the submitted sequences in the NCBI nucleotide database.…”

Sequencing and De Novo Assembly of the Western Tarnished Plant Bug (Lygus hesperus) Transcriptome

“…A total of 7,898 isotigs were assigned GO terms, including 5,961 sequences at the biological process level (Figure 5A), 4,413 sequences at the cellular component level (Figure 5B), and 6,445 sequences at the molecular function level (Figure 5C). The distribution of GO terms within the ontology categories is consistent with other insect transcriptomes [30]–[32], [36]. Within the Biological Process GO category, isotigs assigned to cellular (4,841) and metabolic processes (4,077) were most abundant (Figure 5A).…”

“…For Molecular Function, isotig sequences were predominantly assigned to catalytic activity (4,121) and binding (3,765) functions (Figure 5C). Intriguingly, the number of L. hesperus isotigs assigned with putative antioxidant activity (n = 36) within the Molecular Function GO category, was higher as a percentage (0.56%) than that found in other insects (0.02–0.4%) [22], [30]–[32], [36]. As oxidative damage has been linked to thermal stress [37], the increased expression of antioxidant-related transcripts in L. hesperus may reflect a genetic mechanism that provides some measure of thermotolerance to the extreme climatic conditions of the arid southwestern US.…”

Characterizing the developmental transcriptome of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae) through comparative genomic analysis with Drosophila melanogaster utilizing modENCODE datasets

“…While many P450 proteins are known to be involved in detoxification, the superfamily consists of a wide array of enzymes with distinct functions [17, 18]. This is not the first report of characterization of P450’s in B. dorsalis , with previous research reporting as many as 90 unigenes to as few as 51 from shotgun transcriptome experiments [10, 12, 19]. Inconsistent database deposition of sequences obtained in previous reports makes it difficult to make comparisons and draw conclusions across the respective studies.…”