Differentially expressed microRNAs associated with changes of transcript levels in detoxification pathways and DDT-resistance in the Drosophila melanogaster strain 91-R

Abstract

Dichloro-diphenyl-trichloroethane (DDT) resistance among arthropod species is a model for understanding the molecular adaptations in response to insecticide exposures. Previous studies reported that DDT resistance may involve one or multiple detoxification genes, such as cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), esterases, and ATP binding cassette (ABC) transporters, or changes in the voltage-sensitive sodium channel. However, the possible involvement of microRNAs (miRNAs) in the post-transcriptional regulation of genes associated with DDT resistance in the Drosophila melanogaster strain 91-R remains poorly understood. In this study, the majority of the resulting miRNAs discovered in small RNA libraries from 91-R and the susceptible control strain, 91-C, ranged from 16-25 nt, and contained 163 precursors and 256 mature forms of previously-known miRNAs along with 17 putative novel miRNAs. Quantitative analyses predicted the differential expression of ten miRNAs between 91-R and 91-C, and, based on Gene Ontology and pathway analysis, these ten miRNAs putatively target transcripts encoding proteins involved in detoxification mechanisms. RT-qPCR validated an inverse correlation between levels of differentially-expressed miRNAs and their putatively targeted transcripts, which implies a role of these miRNAs in the differential regulation of detoxification pathways in 91-R compared to 91-C. This study provides evidence associating the differential expression of miRNAs in response to multigenerational DDT selection in Drosophila melanogaster and provides important clues for understanding the possible roles of miRNAs in mediating insecticide resistance traits.

Fig 1. RT-qPCR validation of differentially expressed known and novel miRNAs identified between 91-R and 91-C strains.

The expression levels were normalized by both U6 and 5S rRNA. Statistical significance was analyzed using one-way ANOVA. The asterisks represent significance, where one asterisk indicates P < 0.05.

Fig 2. Gene ontology (GO) analysis and pathway annotation of the most targeted genes of the known differentially expressed miRNAs between 91-R and 91-C strains.

(A) GO analysis. 664 target genes from miRNAs predicted to be differentially expressed were analyzed with FlyMine to obtain the GO terms, and the GO terms were classified with CateGOrizer and separated into three major categories. (B) Pathway annotations. 664 target genes from miRNAs predicted to be differentially expressed were submitted to FlyMine to get the pathway classification. The 30 most abundant pathways were represented.

Fig 3. Putative xenobiotic metabolism-related target genes of differentially expressed miRNAs.

The network consists of differentially expressed miRNAs and their corresponding target genes. Red hexagons represent differentially expressed miRNAs; blue circles represent target genes as phase I detoxification genes; green circles represent target genes encoding phase II detoxification genes; yellow circle represents target genes as phase III detoxification gene; and purple circles represent other genes affecting the insecticide insensitivity in insects. The network was generated and visualized in Cytoscape v3.6.0. Cyp6a8, Cyp6g1, Cyp6g2, and Cyp6w1 associated with DDT resistance are in bold.

Fig 4. Expression level analysis of potential target detoxification genes of differentially expressed miRNAs between 91-R and 91-C strains.

The expression levels were normalized by rp49. Statistical significance was analyzed using one-way ANOVA. The asterisks represent significance, with a single asterisk indicating P < 0.05.