Molecular implications of prolonged DDT selection in Drosophila melanogaster

From the advent of the use of synthetic insecticide in the first half of the twentieth century, insecticide pressure has played a critical role in the evolution of insect populations. Genome-wide monitoring for the effect of such selection has not only provided considerable insight into an understanding of these evolutionary dynamics but also as a way to elucidate changes in individual genes or gene families. Although prior comparative genetic and genomics studies have revealed a large number of individual genes and gene families that may be selectively advantageous for insects over evolutionary time scales, most studies have been limited in that the researchers did not have access to populations within a known common origin for which a given evolutionary selection pressure has been well-defined or documented. In this study, I analyzed the genomes and transcriptomes of a highly DDT-resistant (91-R) Drosophila melanogaster and its corresponding DDT-susceptible (91-C) strain in order to characterize the impact of prolonged DDT selective pressure on genetic diversity for those strains. Comparing the transcriptomic response to DDT in 91-R and 91-C, I identified a multigenic adaptive response to DDT in 91-R along with multiple roles for gene regulatory cascades around stress responses, cell survival, and neurological function. Furthermore, both the genotypic and phenotypic diversity of the phase I detoxification gene, cytochrome P450, as well as phase III detoxification gene, ABC transporter, suggest an adaptive evolutionary response to multigenerational DDT selection pressure between 91-R and 91-C. Taken together, these findings offer evidence for the impact of widespread insecticide selection pressure on genomic diversity in Drosophila evolution along with insights across the entire genome into the functional implications of such positive selection pressure

Impacts of Sub-lethal DDT Exposures on microRNA and Putative Target Transcript Expression in DDT Resistant and Susceptible Drosophila melanogaster Strains

Ten constitutively differentially expressed miRNAs were previously described between DDT-resistant 91-R and -susceptible control Drosophila melanogaster strains, and among their predicted target genes were those associated with metabolic DDT resistance mechanisms. The present study evaluated the inducibility of miRNA expression and putative downstream regulation of cytochrome P450s in response to DDT exposure in a time-dependent manner in 91-R and the susceptible Canton-S strain. Specifically, RT-qPCR analysis showed that DDT exposures led to the significant down-regulation (repression) of miR-310-3p, miR-311-3p, miR-312-3p, miR-313-3p, and miR-92a-3p levels in Canton-S. This is contrasted with the lack of significant changes in 91-R at most time-points following DDT exposure. The levels of expression among miRNAs exhibited opposite expression patterns compared to their corresponding putative target cytochrome P450s at the same time points after DDT exposure. Collectively, results from this study suggest that miR-310-3p, miR-311-3p, miR-312-3p, miR-313-3p, and miR-92a-3p might have a potential role in the control of DDT detoxification through the post-transcriptional regulation of target cytochrome P450s in Canton-S. Conversely, the lack of significant changes of these same miRNAs in 91-R following DDT-exposure suggests a possible adaptive mutation that removes repressive control mechanisms. These data are important for the understanding impact of adaptive changes in miRNA expression on post-transcriptional regulatory mechanism involved in the evolution of DDT resistance in 91-R

Comparative CYP-omic analysis between the DDT-susceptible and -resistant Drosophila melanogaster strains 91-C and 91-R

BACKGROUND: Cytochrome P450 monooxygenases (P450s) are involved in the biosynthesis of endogenous intracellular compounds and the metabolism of xenobiotics, including chemical insecticides.We investigated the structural and expression level variance across all P450 geneswith respect to the evolution of insecticide resistance under multigenerational dichlorodiphenyltrichloroethane (DDT) selection. RESULTS: RNA-sequencing (RNA-seq) and reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) indicated that the transcript levels of seven P450 genes were significantly up-regulated and three P450 genes were down-regulated in the DDT-resistant strain 91-R, as compared to the control strain 91-C. The overexpression of Cyp6g1 was associated with the presence of an Accord and an HMS-Beagle element insertion in the 5′ upstreamregion in conjunction with copy number variation in the 91-R strain, but not in the 91-C strain. A total of 122 (50.2%) fixed nonsynonymous (amino acid-changing) mutationswere found between 91-C and 91-R, and 20 (8.2%) resulted in amino acid changes within functional domains. Three P450 proteins were truncated as a result of premature stop codons and fixed between strains. CONCLUSION: Our results demonstrate that a combination of changes in P450 protein-coding regions and transcript levels are possibly associated with DDT resistance, and thereby suggest that selection for variant function may occur within this gene family in response to chronic DDT exposure

The insulin signaling pathway in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e: A nexus revealing an Achilles\u27 heel in DDT resistance

Insecticide resistance is an ongoing challenge in agriculture and disease vector control. Here, we demonstrate a novel strategy to attenuate resistance. We used genomics tools to target fundamental energy-associated pathways and identified a potential “Achilles\u27 heel” for resistance, a resistance-associated protein that, upon inhibition, results in a substantial loss in the resistance phenotype. Specifically, we compared the gene expression profiles and structural variations of the insulin/insulin-like growth factor signaling (IIS) pathway genes in DDT-susceptible (91-C) and -resistant (91-R) Drosophila melanogaster (Drosophila) strains. A total of eight and seven IIS transcripts were up- and down-regulated, respectively, in 91-R compared to 91-C. A total of 114 nonsynonymous mutations were observed between 91-C and 91-R, of which 51.8% were fixed. Among the differentially expressed transcripts, phosphoenolpyruvate carboxykinase (PEPCK), down-regulated in 91-R, encoded the greatest number of amino acid changes, prompting us to perform PEPCK inhibitor–pesticide exposure bioassays. The inhibitor of PEPCK, hydrazine sulfate, resulted in a 161- to 218-fold decrease in the DDT resistance phenotype (91-R) and more than a 4- to 5-fold increase in susceptibility in 91-C. A second target protein, Glycogen synthase kinase 3β (GSK3β-PO), had one amino acid difference between 91-C and 91-R, and the corresponding transcript was also down-regulated in 91-R. A GSK3β-PO inhibitor, lithium chloride, likewise reduced the resistance but to a lesser extent than did hydrazine sulfate for PEPCK. We demonstrate the potential role of IIS genes in DDT resistance and the potential discovery of an “Achilles\u27 heel” against pesticide resistance in this pathway

Selective Sweep Analysis in the Genomes of the 91-R and 91-C Drosophila melanogaster Strains Reveals Few of the ‘Usual Suspects’ in Dichlorodiphenyltrichloroethane (DDT) Resistance

Adaptation of insect phenotypes for survival after exposure to xenobiotics can result from selection at multiple loci with additive genetic effects. To the authors’ knowledge, no selective sweep analysis has been performed to identify such loci in highly dichlorodiphenyltrichloroethane (DDT) resistant insects. Here we compared a highly DDT resistant phenotype in the Drosophila melanogaster (Drosophila) 91-R strain to the DDT susceptible 91-C strain, both of common origin. Whole genome re-sequencing data from pools of individuals was generated separately for 91-R and 91-C, and mapped to the reference Drosophila genome assembly (v. 5.72). Thirteen major and three minor effect chromosome intervals with reduced nucleotide diversity (π) were identified only in the 91-R population. Estimates of Tajima\u27s D (D) showed corresponding evidence of directional selection in these same genome regions of 91-R, however, no similar reductions in π or D estimates were detected in 91-C. An overabundance of non-synonymous proteins coding to synonymous changes were identified in putative open reading frames associated with 91-R. Except for NinaC and Cyp4g1, none of the identified genes were the ‘usual suspects’ previously observed to be associated with DDT resistance. Additionally, up-regulated ATP-binding cassette transporters have been previously associated with DDT resistance; however, here we identified a structurally altered MDR49 candidate resistance gene. The remaining fourteen genes have not previously been shown to be associated with DDT resistance. These results suggest hitherto unknown mechanisms of DDT resistance, most of which have been overlooked in previous transcriptional studies, with some genes having orthologs in mammals

Dietary Antioxidants Impact DDT Resistance in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e

Insects experience a diversity of subtoxic and/or toxic xenobiotics through exposure to pesticides and, in the case of herbivorous insects, through plant defensive compounds in their diets. Many insects are also concurrently exposed to antioxidants in their diets. The impact of dietary antioxidants on the toxicity of xenobiotics in insects is not well understood, in part due to the challenge of developing appropriate systems in which doses and exposure times (of both the antioxidants and the xenobiotics) can be controlled and outcomes can be easily measured. However, in Drosophila melanogaster, a well-established insect model system, both dietary factors and pesticide exposure can be easily controlled. Additionally, the mode of action and xenobiotic metabolism of dichlorodiphenyltrichloroethane (DDT), a highly persistent neurotoxic organochlorine insecticide that is detected widely in the environment, have been well studied in DDT-susceptible and -resistant strains. Using a glass-vial bioassay system with blue diet as the food source, seven compounds with known antioxidant effects (ascorbic acid, β-carotene, glutathione, α-lipoic acid, melatonin, minocycline, and serotonin) were orally tested for their impact on DDT toxicity across three strains of D. melanogaster: one highly susceptible to DDT (Canton-S), one mildly susceptible (91-C), and one highly resistant (91-R). Three of the antioxidants (serotonin, ascorbic acid, and β-carotene) significantly impacted the toxicity of DDT in one or more strains. Fly strain and gender, antioxidant type, and antioxidant dose all affected the relative toxicity of DDT. Our work demonstrates that dietary antioxidants can potentially alter the toxicity of a xenobiotic in an insect population

Dietary antioxidant vitamin C influences the evolutionary path of insecticide resistance in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e

Herbivorous insects encounter a variety of toxic environmental substances ranging from ingested plant defensive compounds to human-introduced insecticidal agents. Dietary antioxidants are known to reduce the negative physiological impacts of toxins in mammalian systems through amelioration of reactive oxygen-related cellular damage. The analogous impacts to insects caused by multigenerational exposure to pesticides and the effects on adaptive responses within insect populations, however, are currently unknown. To address these research gaps, we used Drosophila as a model system to explore adaptive phenotypic responses to acute dichlorodiphenyltrichloroethane (DDT) exposure in the presence of the dietary antioxidant vitamin C and to examine the structural genomic consequences of this exposure. DDT resistance increased significantly among four replicates exposed to a low concentration of DDT for 10 generations. In contrast, dietary intake of vitamin C significantly reduced DDT resistance after mutigenerational exposure to the same concentration of DDT. As to the genomic consequences, no significant differences were predicted in overall nucleotide substitution rates across the genome between any of the treatments. Despite this, replicates exposed to a low concentration of DDT without vitamin C showed the highest number of synonymous and non-synonymous variants (3196 in total), followed by the DDT plus vitamin C (1174 in total), and vitamin C alone (728 in total) treatments. This study demonstrates the potential role of diet (specifically, antioxidant intake) on adaptive genome responses, and thus on the evolution of pesticide resistance within insect populations

Post-transcriptional modulation of cytochrome P450s, Cyp6g1 and Cyp6g2, by miR-310s cluster is associated with DDT-resistant Drosophila melanogaster strain 91-R

Abstract The role of miRNAs in mediating insecticide resistance remains largely unknown, even for the model species Drosophila melanogaster. Building on prior research, this study used microinjection of synthetic miR-310s mimics into DDT-resistant 91-R flies and observed both a significant transcriptional repression of computationally-predicted endogenous target P450 detoxification genes, Cyp6g1 and Cyp6g2, and also a concomitant increase in DDT susceptibility. Additionally, co-transfection of D. melanogaster S2 cells with dual luciferase reporter constructs validated predictions that miR-310s bind to target binding sites in the 3ʹ untranslated regions (3ʹ-UTR) of both Cyp6g1 and Cyp6g2 in vitro. Findings in the current study provide empirical evidence for a link between reduced miRNA expression and an insecticidal resistance phenotype through reduced targeted post-transcriptional suppression of transcripts encoding proteins involved in xenobiotic detoxification. These insights are important for understanding the breadth of adaptive molecular changes that have contributed to the evolution of DDT resistance in D. melanogaster

Comparative CYP-omic analysis between the DDT-susceptible and -resistant Drosophila melanogaster strains 91-C and 91-R

BACKGROUND: Cytochrome P450 monooxygenases (P450s) are involved in the biosynthesis of endogenous intracellular compounds and the metabolism of xenobiotics, including chemical insecticides.We investigated the structural and expression level variance across all P450 geneswith respect to the evolution of insecticide resistance under multigenerational dichlorodiphenyltrichloroethane (DDT) selection. RESULTS: RNA-sequencing (RNA-seq) and reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) indicated that the transcript levels of seven P450 genes were significantly up-regulated and three P450 genes were down-regulated in the DDT-resistant strain 91-R, as compared to the control strain 91-C. The overexpression of Cyp6g1 was associated with the presence of an Accord and an HMS-Beagle element insertion in the 5′ upstreamregion in conjunction with copy number variation in the 91-R strain, but not in the 91-C strain. A total of 122 (50.2%) fixed nonsynonymous (amino acid-changing) mutationswere found between 91-C and 91-R, and 20 (8.2%) resulted in amino acid changes within functional domains. Three P450 proteins were truncated as a result of premature stop codons and fixed between strains. CONCLUSION: Our results demonstrate that a combination of changes in P450 protein-coding regions and transcript levels are possibly associated with DDT resistance, and thereby suggest that selection for variant function may occur within this gene family in response to chronic DDT exposure

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

<p>(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.</p