Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Entomology
Non Technical Summary
This program will use advanced genomics tools to understand how insect evolve resistance to insecticides, using the fruitfly as a model system. We will study a highly pesticide resistant fly population and compare it to an insecticide susceptble fly population. The outcomes of this project will help guide future studies on insecticide resistance in insect pests that impact crops and human health.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
One major aspect of my program involves the use of molecular biology and genomic tools to elucidate the molecular mechanisms of pesticide insecticide resistance using Drosophila melanogaster (hereafter referred to as Drosophila) as a model organism. The molecular mechanisms that we work on have broad implications for pest species, including Drosophila suzukii, an important pest in Michigan on fruit crops. Briefly, we have developed databases of genomic and transcriptomic experiments from pesticide-susceptible and -resistant Drosophila that allow us to perform multiple "deep dive" studies on both the molecular basis of pesticide resistance and the genomic implications on pest genomes as they relate to evolutionarily conserved pathways. These datasets allow us to perform single gene or pathway analyses that are critical for us to determine candidate genes that can then be tested for biological functions using transgenic Drosophila. For example, our program has and will continue to focus on the molecular changes associated with Phase I, II and III detoxification systems and their implications for understanding the molecular basis of pesticide resistance. In order to perform such "deep dive" studies, we work with a unique pair of fly lines, one DDT-resistant and a DDT-susceptible strain of common origin with the resistant strain. This DDT-Drosophila model system is useful for elucidating fundamental molecular aspects of pesticide resistance that can be potentially applied to economically important pests in order to: (1) understand the evolution of Phase I, II, and III systems, (2) determine the impact of pesticides on evolutionarily conserved genes and pathways, and (2) develop or test the tools to further understand the relative roles of genes (in the aforementioned groups 1 and 2) on pesticide resistance and the possible "costs of resistance".The Drosophila strains that we have are 91-C and 91-R, which originated from a common population taken from the field in the 1950s. This original population was split into two populations: (1) one that was not selected for DDT resistance (91-C) and (2) one that had been selected for multiple decades to high levels of DDT resistance (91-R). The 91-R strain is highly pesticide resistance, and this pair of strains taken together represent an excellent model system to understand the molecular mechanisms associated with high level pesticide resistance. Although Drosophila is a model organism, of little direct agricultural importance and DDT is no longer used for agricultural purposes, this system does serve as an approach to better our understanding of the molecular and evolutionary events associated with insecticide resistance as well as a testing ground for the use of modern genomic tools and emerging bioinformatics analysis approaches that can be used to understand pesticide resistance in insect pests of agricultural importance. To this end, the project contributes to the knowledge area(s) of insects affecting plants (211), animal/insect genomes (304), and animal physiological processes (305). By studying the basic evolution of the Phase I, II and III detoxification systems in Drosophila, we expect it to give us important insights into how other closely related species, like D. suzukii, may eventually evolve resistance to the pesticides currently being used to control their populations. Such evolutionary events may be better understood by a direct analysis of the (1) genome, (2) the transcriptome, (3) analysis of genes that have sequence that is less evolutionarily conserved (e.g., detoxification system genes), or (4) highly evolutionarily conserved genes or gene pathways or both, including, but not limited to nervous system or energy metabolism genes.Our long-term objective is to gain a fundamental understanding of polygenic insecticide resistance whereby, beyond the scope of this project, such information may be useful in the control of economically important pest insects. Our three specific objectives are as follows:CYPome Analysis: Towards understanding the molecular basis, we will complete a CYPome analysis (phase I detoxification systems) for both highly pesticide resistant and susceptible lines of Drosophila across the whole of the CYPome (all the cytochrome P450s in a genome). Within the next three years, we expect to publish at least one paper on this topic.Pathway Analysis: We will continue to analyze genomic and transcriptomic data from 91-C and 91-R to investigate pathway analyses, including genes and pathways likely to be evolutionarily conserved across taxa. We expect one paper to be published on this topic during this project cycle.ABC Transporter/MDR Gene Analyses: We will continue to investigate the relative role of ABC transporters in DDT resistance in the 91-R strain. We will achieve this objective using transgenic insects expressing ABC transporter genes cloned from 91-C and 91-R.
Project Methods
Objective 1 - CYPome Analysis91-R and 91-C Drosophila strains. The Drosophila strains 91-C and 91-R were previously developed (Merrell & Underhill, 1956). Both strains have a common origin, but 91-R has been selected and subsequently maintained on constant exposure to DDT for over 60 years. The 91-C is the corresponding non-selected control strain kept side-by-side in the laboratory without any exposure to DDT selection. 91-R and 91-C strains have been maintained in the Pittendrigh Laboratory since 2000. The transcriptome and genomes of these fly lines are available on a server that Dr. Pittendrigh will continue to access at the University of Illinois Urbana-Champaign with his collaborator Dr. Ken Paige.Validation of P450 gene expression with RNA-seq and RT-qPCR. All RNA-seq read data was previously generated from DDT unexposed 91-C and 91-R in triplicate for each and were submitted to the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA) database (accession number: SRX2611754-SRX2611759). Read data will be imported into the CLC Genomic Workbench 9.5 (Qiagen, Valencia, CA, USA) and differential expression of P450 transcripts will be evaluated between technical replicates of 91-C and 91-R. Specifically, the number of reads per kilobase of transcript per million reads (RPKM) will be mapped against the annotated Drosophila genome assembly v.6.07 as a proxy for gene expression. To establish differential expression between the two strains, the false discovery rate (FDR) method will be used to determine the threshold of the p-value. P450 genes with a false discovery rate (FDR ≤ 0.05) and log2 fold-change ≥ 1.0 will be considered putatively differentially expressed.Reverse transcriptase-quantitative PCR (RT-qPCR) will be applied to validate the expression of putative differentially expressed P450 transcripts between 91-C and 91-R. Total RNA will be extracted from three biological replicates, with the cDNA first strand to be synthesized from 1.0 µg of total RNA using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Subsequent RT-qPCR will be performed using a StepOnePlus Real-Time PCR system (Applied Biosystems Inc., Foster City, CA; located in the Pittendrigh lab) according to manufacturer's instructions. The rp49 gene will be used as an internal control. Melting curve analysis and gel electrophoresis of the PCR products will also be conducted to verify that a single gene-specific product will be produced. Relative expression levels and fold change will be analyzed following comparative Ct method (2-??Ct; Schmittgen & Livak, 2008).Detection of single nucleotide polymorphisms (SNPs) and deletions/insertions (DIPs) in 91-C and 91-R. The SNPs and DIPs will be predicted within P450 gene regions in order to identify potential structural mutations and to define any association with pesticide resistance by comparison between 91-C and 91-R. Putative mutations will be detected by separately mapping RNA-seq read data for 91-C and 91-R to the D. melanogaster reference genome assembly v.6.07 using the CLC Genomic Workbench 9.5 (Qiagen, Valencia, CA, USA) "Map Reads to Reference" tool (minimum length fraction 0.9, minimum similarity fraction 0.8, insertion/deletion cost = 3, and mismatch cost = 3). Mapping data will be used to construct a consensus sequence from the 87 P450 genes annotated in assembly v. 6.07 (noise threshold of 0.1 and minimum nucleotide count of 20). The consensus base frequency of mapped reads will be compared to the reference sequence used to call homozygous (p = 1.0) or heterozygote alleles (p < 1.0) within the depth of reads. The derived amino acid sequence from each P450 gene sequence from 91-C and 91-R will be used in a multiple sequence alignment with the corresponding homologous sequence in strain Canton-S using Clustal Omega (EMBL-European Bioinformatics Institute, Cambridge, UK). Putative substrate recognition sites and other protein signature motifs will be identified based on known P450 functional domains.Objective 2 - Pathway AnalysisRNA-seq and estimation of differential gene expression. Overall, ≥ 80.38% RNA-seq read data from replicates of 91-R (n = 3) and 91-C (n = 3), from which 15,831,798 (1.66 %) of the reads have already been trimmed for this analysis, will be aligned to the D. melanogaster genome v.6.07. Estimates of differential expression will be based on the normalized counts of RNA-seq read data mapped to gene models in order to predict the total number of differentially-expressed transcripts at the low-stringency, with up- and down-regulated genes in 91-R as compared to 91-C. Estimates of fold-change between transcripts from 91-R and 91-C will be correlated between the qRT-PCR and RNA-seq methods.Ontologies, pathways, and interactions among differentially expressed genes (DEGs). The terms for molecular functions, as well as their biological processes, and cellular component categories will be assigned to DEGs. Gene Ontology (GO) categories will be used to reveal which groups or pathways of genes are highly represented among gene products from both up- and down-regulated DEGs. Protein-protein interactions derived from prior two-hybrid screens within Flymine identified experimental lines of evidence to be used to investigate these DEGs. Also, we will investigate connectivity levels via network analyses between up- and down-regulated genes.Objective 3 - ABC transporter/MDR gene AnalysesGeneration of transgenic lines. We will use the same four transgenic fly strains previously used by Seong et al. (2016). These transgenic fly strains contain ORFs as follows: (1) MDR49A from 91-R (91-R-MDR49A); (2) MDR49B from 91-R (91-R-MDR49B); (3) MDR49A from 91-C (91-C-MDR49A); and (4) MDR49B from 91-C (91-C-MDR49B). The mortality bioassay in this case used female progeny from the cross between the MDR49A and B-GAL4 males and female drivers strain (to be deteremined) obtained from Bloomington Drosophila Stock Center (Bloomington, IL). We have also generated fly lines as described by (Seong et al., 2016) in order to generate transgenic flies expressing MDR50 and MDR65, previously implicated in DDT resistance in the 91-R fly line.Bioassay with topical applications. In order to understand the potential role of ABC transporters in cross resistance bioassays, we will use seven technical-grade insecticides representing different classes: DDT, abamectin, carbofuran, imidacloprid, methoxychlor, permethrin, and prothiofos (Sigma-Aldrich, St. Louis, MO, USA). Females from F1 that transgenically express MDR49A and MDR49B from 91-R and 91-C will be selected as the experimental group for mortality bioassays. Insecticide toxicity for the four transgenic Drosophila strains will then be assessed by topical applications using the following procedure. A stock solution for each insecticide will be prepared and diluted in acetone to a series of concentrations suitable for bioassays. Subsequently, 0.2 µl will be applied to the pronotum of five-day-old females using a handheld microapplicator (Hamilton Co., Reno, NV, USA). Insecticide-treated flies will be transferred to vials capped with cotton plugs moistened with a 5% sucrose solution in distilled water. For each insecticide, five doses with thirty flies per dose will be biologically repeated three times on different days. An acetone-only treatment will be included in each replicate as a negative control. Mortality will be assessed at 24 hours post-treatment. Flies will be considered dead when all movement and leg twitching had ceased. Mortality rates will then be analyzed by Probit (XLSTAT 2008, Addinsoft, USA). The 50% lethal dose (LD50), 95% confidence intervals, and slopes and intercepts for dose-response curves will be generated to determine the dose of topically applied insecticide.