Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Mol. & Environ. Toxicology
Non Technical Summary
The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a major agricultural pest that threatens the security of potato crops across the United States. Imidacloprid, a neonicotinoid insecticide, is commonly used for control of L. decemlineata, but many populations of L. decemlineata have begun to show increased imidacloprid resistance. Studies have demonstrated that L. decemlineata up-regulate detoxifying enzymes in the presence of insecticides, resulting in varying degrees of resistance in geographically distributed populations. We hypothesize that it is not solely the presence of the insecticides that leads to increased resistance, but rather a combination of multiple factors, including the application of fungicides on potato crops. In this study we seek to uncover whether the use of fungicides leads to cross- resistance to insecticides and may have an additive effect. With the proposed USDA AFRI ELI postdoctoral fellowship, we will determine the effects of fungicidal exposure on L. decemlineata and whether exposure to fungicides leads to increased imidacloprid resistance, whether fungicidal exposure leads to overexpression of genes that are up-regulated in response to the imidacloprid, and the correlation between geographic distribution of resistance and the presence of fungicides. Completion of the research objectives will advance the sustainability of crop production by revealing the underlying causes which lead to imidacloprid resistance in L. decemlineata and will reduce the need for increasing pesticide application. Controlling pesticide resistance in L. decemlineata is vital to ensure the economics and security of potatoes, as well as limit pesticide release into the surrounding environment.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
0%
Goals / Objectives
GOALSThe proposed research will broaden our understanding of potential drivers of insecticide resistance. Specifically, we will uncover the environmental interactions of insecticides and fungicides which may lead to increased rates of pesticide resistance in the Colorado potato beetle (Leptinotarsa decemlineata (Say)). The current proposal will test the novel idea that xenobiotic compounds (fungicides) influence the rates of resistance evolution in this important species and provide far-reaching impact on insect resistance management strategies that currently do not take into account off-target effects of non-insecticidal pesticide treatments. The proposed research examines this possibility and aids in our ability to determine if fungicides are activating detoxification cascades and associated genes that lead to insecticide resistance in this invasive species.Exploration into insecticides and fungicides cross resistance will assist in maintaining the long-term sustainability of the potato agricultural system. The information and technology discovered during this fellowship may lead to an improved understanding of control measures for insecticide resistant populations of beetles while reducing, or slowing insecticide resistance. Reduced resistance, in turn, could decrease insecticide applications required for pest control, which may itself be a human health hazard, and is certainly an environmental impact. Further, this study would provide evidence that other chemicals introduced into agricultural systems need to be considered in management of insecticide resistance. Finally, the findings in this study could potentially lead to new technologies for pest control, such as the discovery of plausible targets (up-regulated genes) for small interfering RNAs, which would increase the overall efficiency of agricultural production through a reduction in total insecticide inputs and resistance development.OBJECTIVESDetermine the impact of exposure to commonly used fungicides on the susceptibility of select populations of L. decemlineata to imidacloprid, a commonly used neonicotinoid insecticide.Determine the concentrations of commonly used fungicides which are lethal to L. decemlineata.Determine whether exposure to sub-lethal concentrations of fungicides increases the LC50 of imidacloprid in L. decemlineata.Identify genes that are commonly up-regulated in imidacloprid susceptible populations of L. decemlineata following fungicide or imidacloprid exposure.Determine whether fungicide and insecticide susceptibility varies between different geographic populations of L. decemlineata and whether genes which are up-regulated after fungicide exposure in imidacloprid susceptible populations are constitutively up-regulated in either population.
Project Methods
Objective 1: We will obtain a lab strain of imidacloprid susceptible L. decemlineata. The lab-susceptible population of L. decemlineata will be maintained in an insecticide-free greenhouse at the UW-Madison. Fungicides used in this study will include: flutolanil, dithiocarbamate, chlorothalonil, and boscalid, the most widely used fungicides in the US.We will conduct LC50 assays to examine the lethal concentration of flutolanil, dithiocarbamate, chlorothalonil, and boscalid upon an imidacloprid susceptible beetle population. The results of this objective will determine whether field application of each fungicide has a negative impact on beetle fitness, as well as determining the lethal dose of each fungicide in L. decemlineata. Using a topical bioassay, we will test a range of concentrations of the individual fungicides on the susceptible beetle population and determine the LC50 of each fungicide. Using serial dilutions of technical fungicides in acetone, we will dose 350 adult beetles per fungicide. Beetles will be divided into 7 groups (n=50) for a serial dilution of fungicide concentrations and treated with 1µl of solution on the first abdominal sternite. A wide range of fungicide doses will be initially screened to better determine an appropriate set of serial dilutes for LC50 studies. A 0 ppm concentration in acetone will be used as a no fungicide control. Seven days after pesticide application, the number of live beetles, incapacitated beetles, and dead beetles will be recorded, as measured by the pencil test to determine an estimated LC50. Briefly, adult beetles will be presented with the opportunity to climb a pencil: if they can move a full body length they will be considered alive, if they appeared alive but cannot move a body length they are considered incapacitated, and if there is no movement, even after pinching the back legs, they will be considered dead. Abbott's correction will be run to normalize the data and a log10 probit regression analysis (PROC PROBIT, SAS Institutes) will be used to estimate the LC50..Adult beetles will be divided into 5 groups (n=350 per group) and dosed with 1ml of a sublethal concentration of each of the 4 selected fungicides in acetone or the acetone control. A LC50 assay, as described for imidacloprid, will be conducted 24 hours after fungicide exposure to determine whether exposure to each fungicide increases the LC50 of imidacloprid in the susceptible L. decemlineata population. In each fungicide treatment, beetles will be further divided into 7 groups (n=50) for a serial dilution of imidacloprid concentrations and treated with a 1µl solution of imidacloprid suspended in acetone on the first abdominal sternite. A 0 ppm imidacloprid in acetone concentration will be used as a control. Seven days after pesticide application, the number of live beetles, incapacitated beetles, and dead beetles will be recorded. From this data, we will determine if prior fungicide application increases the resistance of L. decemlineata to imidacloprid.Objective 2: An imidacloprid susceptible lab strain will be used to examine gene regulation after imidacloprid or fungicide exposure. We will identify genes that are commonly up-regulated in imidacloprid susceptible populations of L. decemlineata following fungicide or imidacloprid exposure. An imidacloprid susceptible lab strain will be used to examine gene regulation after imidacloprid or fungicide exposure. We will determine candidate genes which are up-regulated in imidacloprid resistant L. decemlineata compared to imidacloprid susceptible populations and are involved in xenobiotic detoxification. Using preexisting literature and RNA-sequencing data for imidacloprid resistant and susceptible populations of L. decemlineata, we will determine genes which are up-regulated in imidacloprid resistant populations and are involved in xenobiotic metabolism for further investigation. Previous studies have classified possible mechanisms of resistance between imidacloprid susceptible and resistant populations through transcriptome construction and analysis of gene expression.Next, we will determine whether the candidate genes identified previously are up- regulated in imidacloprid susceptible L. decemlineata after exposure to the fungicides which were found to cause cross-resistance in Objective 1 or a mixture of imidacloprid and fungicides. We will develop a panel of qPCR primers to compare activation of identified candidate genes after imidacloprid or fungicide exposure. Although it is possible we may have many candidate genes, we will limit the number of genes that we investigate to the top 5-10 candidates previously described as important for insecticide resistance. We will dose beetles with either a non-lethal concentration of fungicide, a non-lethal dose of imidacloprid, or both to induce gene expression. These concentrations will be determined using the LC50 data from Objective 1. We will then extract RNA from the beetles to examine gene regulation. We will extract RNA from 60 individuals for each chemical inducer.Total RNA will be treated with TurboDNase and the quality of the RNA will be examined. DNA contamination will be removed and DNA-free, RNA can be purified to remove any possibility of PCR inhibition. Following purification, RNA will be suspended in 20µl of water, and the integrity will be examined. All RNA concentrations shall be equalized before input into the cDNA synthesis kit and the subsequent cDNA will be generated with a high capacity cDNA reverse transcription kit. The cDNA will be diluted to a final concentration of 5ng/µl of RNA equivalent input for qPCR. β-actin will be used as a reference gene. The qPCR reaction will be run with a master mix of Bullseye EverGreen. Transcriptional changes will then be quantified and compared between the imidacloprid, fungicide, and mixture exposed beetles.Objective 3:.Populations of L. decemlineata from different geographic regions in the US vary in their resistance to insecticides. These unique populations will be obtained representing western populations and compared to Eastern and Midwestern populations. Our approach to complete this part of the study will be to acquire beetle populations from each unique geographic region of the US through our collaborators. We will determine the lethal concentration of select fungicides and the insecticide imidacloprid for these populations with the procedure previously described. We will then measure gene response to selected fungicides by determining gene expression from our qPCR panel, as described in Objective 2, following fungicide exposure compared to a no fungicide exposure control treatment.Evaluation: We will evaluate the impact of our research by examining the impact of the journal articles which result from our studies, through changes in pest management recommendations for insecticide resistance prevention and strategies for managing resistant populations, and through the development of potential products for the targets (up-regulated genes) our studies uncover, including small interfering RNAs. These impacts will increase the overall efficiency of agricultural production through a reduction in total insecticide inputs and resistance development.