TESTING THE POLYGENIC BASIS OF INSECTICIDE RESISTANCE IN COLORADO POTATO BEETLE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013067
• Project Start Date: Oct 1, 2017
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Project Director
Schoville, SE, DE.

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
Entomology

Non Technical Summary
Pesticide resistance remains an important problem in agriculture, as it limits productivity, incurs financial costs, and requires unsustainable levels of chemical inputs. The ability of pests to develop resistance is still poorly understood, particularly regarding the process of adaptive change that leads to resistance. Understanding the mechanisms involved in pesticide adaptation is important because resistance management is expected to be durable only when simple (single-gene) mutations cause resistance, and less so when multiple loci and molecular pathways confer resistance. It is questionable whether single genes adequately explain most cases of insecticide resistance, and despite early studies arguing that polygenic (multiple-gene) evolution from standing (ancestral) variation is likely to be the norm, most research continues to focus on de novo mutational change as the source of adaptive variation. Polygenic resistance drawn from standing variation could explain how insects rapidly overcome multiple classes of pesticides. The purpose of this project is to apply novel genomic approaches to test for polygenic insecticide resistance and the role of standing variation in the Colorado potato beetle (CPB), Leptinotarsa decemlineata. This proposal addresses the Wisconsin state priority to understand "Mechanisms of pest and pathogen resistance and safe and effective control." Within the USDA National priority to reduce insect damage and improve crop health and production globally, the research addresses subject areas including "insect population and molecular genetics," "identifying mechanisms of pest resistance to control methods," and "developing safe and effective pest control."

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3110	1080	75%
211	3110	1150	25%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1080 - Genetics; 1150 - Toxicology;

Keywords

insecticide resistance

insect pest

colorado potato beetle

rapid evolution

genome sequencing

adaptation

genetic mapping

Goals / Objectives
Pesticide resistance remains an important problem in agriculture, as it limits productivity, incurs financial costs, and requires unsustainable levels of chemical inputs. The ability of pests to develop resistance is still poorly understood, particularly regarding the process of adaptive change that leads to resistance. Understanding the molecular mechanisms underlying pesticide adaptation has become increasingly important because of the widespread occurrence of the "pesticide treadmill" phenomenon in agricultural pests, wherein the repeated and escalating use of pesticides, as well as the faster introduction of new chemistries, is required to keep pace with pest evolution. The pesticide treadmill is obvious, when, for example, the history of pesticide introduction and rate of pest evolution is examined for the top insect pests on the Insecticide Resistance Action Committee database. Colorado potato beetle (CPB),Leptinotarsa decemlineata, is among the most notorious, with documented resistance to 56 insecticides spanning all modes of action. From early insecticides such as Paris Green to DDT, to modern insecticides such as pyrethroids and neonicotinoids, CPB populations have amassed resistance over impressively short periods of time, even within one year of the introduction of novel chemicals. A fundamental question with important implications for resistance management is whether the same genes and molecular pathways repeatedly underlie CPB's ability to adapt to a broad range of insecticides? Answering this question would allow us to develop better predictive models of resistance evolution and to avoid similar pitfalls with the introduction of new chemical control methods.At a more fundamental level, this question requires understanding the mechanisms that underlie rapid genetic adaptation, a highly debated topic in evolutionary biology. Under one mechanism, species rapidly evolve by drawing upon standing (ancestral) genetic variation. An alternative mechanism is that species adapt via selection on de novo mutation(s). Most evolutionary biologists would agree that a combination of both evolutionary mechanisms probably occur in many cases of adaptation, as it is evident that phenotypic traits are typically quantitative in nature (influenced by multiple loci) and the most likely outcome of evolution in theoretical models is polygenic adaptation. However, identifying the role of polygenic evolution from standing variation can be a difficult process. The purpose of this project is to apply novel genomic approaches to test for polygenic insecticide resistance and the role of standing variation in the Colorado potato beetle (CPB), Leptinotarsa decemlineata.The project has two objectives:1) Employ quantitative trait locus mapping to identify causal loci underlying the neonicotinoid resistance phenotype in Wisconsin, and integrate these results with preliminary data from transcriptome sequencing and genome scans of selection to characterize the genetic networks underlying resistance.2) Use whole genome resequencing and temporal sampling of museum specimens to test for the role of standing genetic variation in the evolution of pesticide resistance in CPB.

Project Methods
Objective 1: Determine if resistance to neonicotinoid pesticides is polygenic 1a. Identify loci underlying the resistance phenotype and their effect size. QTL mapping will be used to measure statistical associations between genetic markers and neonicotinoid resistance in an F1 intercross experiment. If multiple QTL are statistically identified in this experiment, this provides evidence of polygenic evolution. In 2016, male and female beetles from highly resistant and susceptible populations in Wisconsin were reciprocally crossed. Fifty pairs of the male and female F1 siblings were mated to produce F2 full-sibling families. Using F2 full-sib families instead of half-sibs balances a trade-off between maximizing power to detect QTLs above background genetic variation and maximizing the number of potentially informative families. Founding parents, F1 parents, and F2 progeny were phenotyped in fall 2016 for weight, size, and imidacloprid resistance. I plan to genotype founding and F1 parents to identify marker-informative F2 families. At least 10 F2 individuals per informative family will be genotyped, for a total of 500 F2 individuals. Specifically, sequences representing a genome-wide sample of genetic variation will be generated using a genotyping-by-sequencing approach. A genetic linkage map will be constructed and composite interval mapping will be used to identify regions associated with neonicotinoid resistance and to estimate their effect size.1b. Identify gene networks underlying neonicotinoid insecticide resistance. Data from QTL mapping, transcriptome sequencing, and genome scan analyses will be integrated to answer the fundamental question of whether insecticide resistance is polygenic in Wisconsin CPB. Depending on the method employed, selection on polygenic traits can be difficult to detect, as it results in moderate changes in allele frequencies (soft or partial selective sweeps) that are less likely to have the strong significance values. If multiple genes are implicated in neonicotinoid resistance by all three methods, my results will provide support for polygenic evolution. Combining information across multiple approaches provides complementary (and potentially corroborating) data about the genetic loci and pathways underlying resistance phenotypes. One frequent concern in analyses of large datasets is the inclusion of false positive hits (Type I error) resulting from the large number of statistical tests. Normally this is resolved by adjusting p-values to more conservative values, by implementing recalibrations such as the Bonferroni correction. However, in functional genomic studies, significant genes may be more easily identified in follow-up studies than by removing them based on p-value. Given our multiple data sources, test calibration for each dataset can be relaxed using the less conservative false discovery rate to provide the largest lists satisfying a calibrated significance level, because overlap in gene function and pathway among the methods will provide the strongest support for the role of particular genes. Analyses of the significant hits from each experimental approach will be annotated for gene function based on gene ontology (GO) terms and molecular pathway using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Statistical tests of gene function and molecular pathway enrichment will be conducted.Objective 2: Assess the role of de novo mutation and standing genetic variation in CPB insecticide resistance evolution2a. Determine if there are genetic differences in the underlying resistance genes among populations. Genetic variation at the loci involved in resistance should be substantially reduced if it results from de novo mutation, and adaptive alleles should also be geographically limited to pest populations. Spatial sampling of non-pest populations and closely related species will therefore be used to test for the contribution of de novo mutation and standing genetic variation in CPB insecticide resistance. I will analyze existing data from 88 CPB genomes, and 10 genomes from related species, from a broad geographical sampling to account for invasion history. In 6 states (Wisconsin, Michigan, Oregon, Maine, Maryland, and New York), samples (5 each) of neonicotinoid resistant and susceptible beetles will be directly compared for evidence of recent, directional selection on novel mutations. Samples have been sequenced on the Illumina HiSeq2500 as 100 bp pair-end reads (to 7x coverage for CPB, 20x for other species). SNP discovery has been performed using GATK and resulted in more than 76 million SNPs and 10 million insertions/deletions (indels) within CPB. To identify population boundaries, SNP variation will be examined in the program ADMIXTURE, which simultaneously assigns individuals to populations and estimates mixture proportions. To identify recent directional selection in resistant-susceptible population pairs, the allele frequency spectrum of SNP loci and imputed haplotypes in the hapFLK statistic will be used to identify selective sweeps. The programs BAYESCAN and LFMM will be also used to scan genomes for evidence of pesticide selection. For those alleles significantly associated with pesticide resistance, their age will be estimated using an approximate Bayesian computation framework. Finally, we will examine whether indels and genome rearrangements might explain patterns of rapid evolution in CPB by estimating their rate of accumulation in a Bayesian phylogeny, as well as in pair-wise comparisons of populations and closely-related species of Leptinotarsa.2b. Determine if there is a change in allele frequency associated with pesticide selection through time. Alleles linked to pesticide resistance should increase in frequency as a result of selection, so a comparison of modern beetles and beetles that predate pesticide use should exhibit allele frequency differences. Furthermore, if standing genetic variation plays a role in insecticide resistance evolution, alleles associated with the resistance phenotype should be present preceding pesticide usage. Thus, temporal samples provide an opportunity to not only detect selection, but provide a means to directly assess the role of standing variation in resistance evolution. Historical collections of CPB from Wisconsin and nearby regions are available for this experiment. Four pools of beetles from Madison will be compared (2 contemporary, 2 prior to insecticide use), as well as three additional historical samples from nearby regions that will be used to measure the potential influence of gene flow. The contemporary samples will include a neonicotinoid resistant and susceptible population. We will submit individuals from each time point/location for whole-genome resequencing. Museum specimens will be extracted individually in an ancient DNA facility (Molecular Archaeology lab at UW) and sequenced on the Illumina HiSeq2500 platform to obtain 100 bp reads. The filtered reads will be aligned to the CPB reference genome using BWA-MEM followed by SNP discovery in GATK, after masking repeat regions using RepeatMasker. ANGSD will be used to identify allele frequency differences among samples, using a likelihood ratio test to account for uncertainty in the observed genotypes and a false-discovery rate correction.An important focus of the analysis will be on alleles that are rare or absent from old populations (including regional populations that could have contributed alleles via gene flow), which appear to undergo frequency increases in modern samples following episodes of pesticide selection. Following other studies, confidence intervals for the frequency change in alleles will be calculated using non-parametric bootstrap or coalescent simulations. This allows conservative estimates of allele frequency change to ensure that alleles recorded as absent from the old population are not merely from genomic regions where degraded DNA did not map well.

Progress 10/01/17 to 09/30/21

Outputs
Target Audience:1) US potato growers and pest management professionals, 2) scientists investigating insect pest evolution, and 3) the Madison community. • Extension: Our support of potato growers and pest management professionals involved personal farm visits, speaking at extension/grower events, and reporting to cooperating grower associations. We presented talks and posters annually (2017-2022) at the Wisconsin Potato and Vegetable Grower annual Education meeting in February. We sent annual research reports to the Wisconsin Potato and Vegetable Growers. • Research: We worked with the community of researchers investigating insect pest evolution by communicating research findings through presentations at the Arthropod Genomics Symposium and the Entomological Society of America. A planned symposium and talk at the International Congress of Entomology in Finland were postponed until July 2022 due to COVID-19. We published four papers and submitted two additional book chapters. We also held regular meetings with research collaborators working on Colorado potato beetle resistance in Vermont, Maryland, and Nebraska. • Outreach: This research has facilitated the training and engagement of one paid UW undergraduate, as well as a high school student and three additional undergraduates through other financial sources. We developed a K12 outreach game based on insecticide resistance that we have used at Wisconsin Discovery Days in Madison, and in local schools in the Madison Municipal School District. Changes/Problems:Our QTL project has not been finalized. In 2020/2021, we developed a new genome assembly and as a result, we have not completed the QTL analysis. We are currently awaiting publication of the improved genome before we proceed with publishing the QTL study What opportunities for training and professional development has the project provided?The project has provided training opportunities for four undergraduates, two high school students, and training and professional development opportunities for two graduate students. Emma Fischer and Syndey Schumacher are high school students who received training on this project through the Wisconsin Youth Apprenticeship Program. Mary Magnuson worked on this project as an undergraduate and received an Agricultural Research Station Internship award. She is now pursuing graduate school. Zach Cohen received his PhD in 2021 and was awarded the Wisconsin Distinguished Graduate Fellowship award for his work in the project. He is now a postdoctoral scientist at the USDA in Texas. Yi-Ming Weng will receive his PhD in 2022. All students in this project developed experience with data collection, data analysis, public presentations, and scientific writing. How have the results been disseminated to communities of interest?Findings from this research were disseminated to: 1) cooperating Wisconsin potato growers via industry trade group meetings from 2017-2022, 2) vegetable growers in Wisconsin at a grower/Extension conference from 2017-2022, 3) to entomologists at the annual Entomology Society of America meetings and the Arthropod Genomic Symposium meetings from 2017-2021 (this will include a planned international presentation at the International Congress of Entomology in Helsinki, Finland in July 2022), 4) collaborators working on Colorado potato beetle in the US based in Maryland, Vermont and Nebraska, and 5) and students in the Madison Metropolitan School District through Wisconsin Discovery Days and training of one high school intern. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This research identified the genetic mechanisms contributing to the increase of insecticide resistance in Colorado potato beetle (CPB),Leptinotarsa decemlineata, populations in the U.S. Specifically, through multiple experimental approaches leveraging genomics and phenotypic data, this research showed that the same genetic pathways are repeatedly selected in different populations of CPB, suggesting CPB has a broad ability to rapidly evolve to insecticides and resistance does not spread via dispersal. The research data show that CPB has abundant genomic variation, and a redundancy of genes that work in similar genetic pathways, which evolution has shaped into novel resistance phenotypes. The project sought to provide current information on the efficacy of insecticides, particularlywidely-used neonicotinoid insecticides, in protecting potatoes from the Colorado potato beetle (CPB). Importantly, there is a clear pattern of metabolic resistance evolving in different populations, which suggests that CPB has the ability to break-down and excrete not only neonicotinoids, but also a broad spectrum of insecticides. This result suggests there are challenges ahead in mitigating resistance development and novel control measures will be needed. The goal of our first objective was to identify genes and genetic mechanisms underlying insecticide resistance in Wisconsin, as well as other potato growing regions in the U.S. We also developed genomic, transcriptomic, epigenetic, and phenotypic (resistance) data from across the range of CPB, and employed multiple approaches to identify the genetic basis of resistance in different populations. Our key results were as follows: 1) We identified metabolic detoxification networks (CYP, GST, and ABC transporters) as playing a primary role in resistance, and showed that constitutive regulatory changes explained gene expression differences among geographically isolated resistance populations. 2) We showed that genomic diversity is high and repeated selection occurs on similar genetic pathways in different geographical populations of CPB. 3) We identified a large list of candidate insecticide genes that could explain how resistance evolves in CPB. 4) We showed that gene exchange appears to be limited among contemporary CPB populations, even within a growing region (such as the Pacific Northwest or Wisconsin). 5) We showed that CPB, and closely related beetles in the genus Leptinotarsa, appear to have high rates of evolution due to larger effective population sizes, which may explain rapid evolution to a range of environmental conditions, including diverse host plants, novel climates, and agroecosystem management. 6) Finally, we showed that epigenetic sources of variation might contribute to gene expression differences in resistant beetles, as lab-reared beetles showed this pattern in a selection experiment. For our planned QTL experiment, we performed an initial data collection of 2 families resulting from crosses of resistant and susceptible populations of Wisconsin Colorado potato beetles. Phenotyping was conducted, and genomic data were collected. We are still working on analyzing this data. For this portion of the project, we published 12 papers and one is in review. The goal of our second objective was to use genome resequencing and museum sampling to test a specific hypothesis about how standing genetic variation (variation present prior to selection events) could contribute to insecticide resistance evolution. We developed whole genome resequencing data from across the range of Colorado potato beetles, with extensive population sampling in Wisconsin and New York. We also collected genomic DNA from >100 museum specimens, representing populations from Wisconsin and New York, focusing on CPB samples originating prior to industrial pesticide use. Our key results were as follows: 1) While standing genetic variation contributed to convergent patterns of resistance in different geographical regions of the U.S., analyses leveraging large sample sizes in Wisconsin and New York show that new mutations also account for resistance. 2) Resistance genes are unique to highly resistant populations in New York and Wisconsin and include transcription factors that drive metabolic resistance phenotypes. 3) Our museum CPB data suggest that population structure increased, and effective size declined in response to industrial insecticide use. Unfortunately, the data are too low in sequencing coverage and have too much missing data to infer details about selection events at candidate genes. For this portion of the project, we will publish two papers (both are currently in review).

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: P�lissi�, B., Chen, Y.H., Cohen, Z.P., Crossley, M.S., Hawthorne, D.J., Izzo, V. and Schoville, S.D., 2022. Genome resequencing reveals rapid, repeated evolution in the Colorado potato beetle. Molecular biology and evolution Early View
• Type: Book Chapters Status: Published Year Published: 2021 Citation: Schoville, S.D., Z.P. Cohen, and M.S. Crossley. 2021. Population genomic insights into insecticide resistance in the Colorado potato beetle. in J.R. Dupuis and O. Rajora (eds.), Population Genomics. Springer, Cham. https://doi.org/10.1007/13836_2021_91
• Type: Book Chapters Status: Awaiting Publication Year Published: 2022 Citation: Crossley, M.S., Z. Cohen, B. P�lissi�, S. Rondon, Y. Chen, A. Alyokhin, D. Hawthorne, and S.D. Schoville. Limiting a superpest: Ecological and evolutionary factors mitigating Colorado potato beetle (Coleoptera: Chrysomelidae) adaptation to insecticides. in A. Alyokhin and Y. Gao (eds.), Insect Pests of Potato, 2nd ed.
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Cohen, Z. S.D. Schoville, and D.H. Hawthorne. The role of structural variants in pest adaptation and genome evolution of the Colorado potato beetle (Say). Molecular Ecology [Authorea https://doi.org/10.22541/au.163109527.73747035/v1]
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Cohen, Z. and S.D. Schoville. Evidence of hard-selective sweeps suggest independent adaptation to insecticides in Colorado potato beetle (Coleoptera: Chrysomelidae) populations. Evolutionary Applications
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Brevik, K., E. Bueno, S. McKay, S. Schoville, and Y. Chen. 2021. Insecticide exposure affects intergenerational patterns of DNA methylation in the Colorado potato beetle, Leptinotarsa decemlineata. Evolutionary Applications 14(3): 746-757.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Dively, G.P., M.S. Crossley, S.D. Schoville, N. Steinhauer, N., and D. J. Hawthorne. 2020. Regional differences in gene regulation may underlie patterns of sensitivity to novel insecticides in Leptinotarsa decemlineata. Pest Management Science 76(12): 4278-4285.
• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Cohen, Z.P., O. Francois, and S.D. Schoville. "Museum genomics of an agricultural super-pest, the Colorado potato beetle, Leptinotarsa decemlineata." Integrative & Comparative Biology
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Thomas, G.W.C., Dohmen, E., Hughes, D.S.T. et al. Gene content evolution in the arthropods. Genome Biol 21, 15 (2020). https://doi.org/10.1186/s13059-019-1925-7

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:1) US potato growers and pest management professionals, 2) scientists investigating insect pest evolution, and 3) the Madison community. • Extension: Our support of potato growers and pest management professionals continues to involve personal farm visits, speaking at extension/grower conferences, and reporting to cooperating grower associations. We presented talks and posters at the Wisconsin Potato and Vegetable Grower annual Education meeting in February 2020. • Research: We continue to engage with the community of researchers investigating insect pest evolution, communicate research findings through regular meetings with collaborators in Vermont and Maryland, and through presentations at the 2020 Arthropod Genomics Symposium. Planned talks at the International Congress of Entomology in Finland were canceled due to COVID-19. We published four papers and submitted two additional book chapters. • Outreach: This research has facilitated the training and engagement of one UW undergraduate. We continue seeking outlets for communicating the importance of entomology and insect pest evolution. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training opportunities for one undergraduate, and training and professional development opportunities for two PhD candidates, Zach Cohen and Yi-Ming Weng. One undergraduate, Emma Fischer, was involved in laboratory genetics and gene knockdown trials related to this project. Zach Cohen,a PhD candidate, published his first paper relating to this project. How have the results been disseminated to communities of interest?We communicated with the Wisconsin Potato and Vegetable Growers about our research at their annual Extension Grower Education Conference in February 2020 in both a talk and poster. Graduate student Zach Cohen presented the results of his research at the 2020 Arthropod Genomics Symposium. Zach and Sean Schoville planned to present at the 2020 International Congress of Entomology, but it was canceled due to COVID (and rescheduled for 2022). We published four manuscripts, have one book chapter accepted and another book chapter under review. What do you plan to do during the next reporting period to accomplish the goals?Objective 1) We plan to finalize QTL analyses and integrate those into a publication with a new genome assembly for Colorado potato beetle. Objective 2) We will publish our analysis of whole genome resequencing data and the role of standing genetic variation in regional adaptation to insecticides. We will complete analysis of genome sequencing data from museum specimens to complete a temporal analysis of demography in two regions (New York and Wisconsin). Results from the efforts will be presented at the Wisconsin Potato and Vegetable Growers Extension Grower Education Conference in February 2021, and the International Congress of Entomology meeting in Finland in 2022.

Impacts
What was accomplished under these goals? Objective 1) We performed an initial data collection of 2 families resulting from crosses of resistant and susceptible populations of Wisconsin Colorado potato beetles. Phenotyping was conducted, and genomic data were collected. In 2019/2020, we developed a new genome assembly and attempted to constructed a linkage map. However, it was determined that we had insufficient sample size to do this analysis. We are currently waiting for additional sequence data from additional families (there has been a delay due to COVID). We will then proceed to conduct the QTL study. As an alternative, we also developed and analyzed transcriptomic data. One paper utilizing this data have been published. We identified metabolic detoxification networks (CYP, GST, and ABC transporters) as playing a primary role in resistance, and showed that constitutive regulatory changes explained gene expression differences among geographically isolated resistance populations. Objective 2) We completed analyses of whole genome resequencing data from across the range of Colorado potato beetles, identifying signatures of standing genetic variation contributing to convergent patterns of resistance in different geographical regions of the USA. One manuscript is very near submission. We also collected genomic DNA from >250 museum specimens, representing populations from the Midwest and Eastern US, and covering a period of >100 years (including prior to industrial pesticide use). Although we have not yet analyzed the data, it does appear that coverage is quite low and our inferences may focus on demographic history (rather than detailed analyses of selection). We found sample size prohibited generation of a linkage map and related QTL analyses. We are boosting sample size for this analysis. Coverage from the museum sequencing data was lower than expected. Although we are still working with the data, it appears insufficient to address questions about selection, but may instead be quite useful for understanding levels of genetic diversity through time (and related estimated of demographic change).

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Dively, G.P., M.S. Crossley*, S.D. Schoville, N. Steinhauer, N., and D. J. Hawthorne. Accepted, 2020. Regional differences in gene regulation may underlie patterns of sensitivity to novel insecticides in Leptinotarsa decemlineata. Pest Management Science 76(12): 4278-4285.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Brevik, K., E. Bueno, S. McKay, S. Schoville, and Y. Chen. 2020. Insecticide exposure affects intergenerational patterns of DNA methylation in the Colorado potato beetle, Leptinotarsa decemlineata. Evolutionary Applications
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Crossley*, M.S., K.D. Burke, S.D. Schoville, and V.C. Radeloff. 2021. Recent collapse of crop belts and declining diversity of US agriculture since 1840. Global Change Biology 27(1): 151-164.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Cohen*, Z.P., K. Brevik, Y.H. Chen, D.J. Hawthorne, B. Weibel , S.D. Schoville. 2020. Elevated rates of positive selection drive the evolution of pestiferousness in the Colorado potato beetle (Leptinotarsa decemlineata, Say). Molecular Ecology 30(1): 237-254.
• Type: Book Chapters Status: Accepted Year Published: 2021 Citation: Accepted, 2020: Crossley*, M.S., Z. Cohen*, B. P�lissi�*, S. Rondon, Y. Chen, A. Alyokhin, D. Hawthorne, and S.D. Schoville. Limiting a superpest: Ecological and evolutionary factors mitigating Colorado potato beetle (Coleoptera: Chrysomelidae) adaptation to insecticides. in A. Alyokhin and Y. Gao (eds.), Insect Pests of Potato, 2nd ed.
• Type: Book Chapters Status: Under Review Year Published: 2021 Citation: In Revision, 2020: Schoville, S.D., Z.P. Cohen*, and M.S. Crossley*. Population genomic insights into insecticide resistance in the Colorado potato beetle. in J.R. Dupuis and O. Rajora (eds.), Population Genomics: Insects. Springer.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:1) US potato growers and pest management professionals, 2) scientists investigating insect pest evolution, and 3) the Madison community. • Extension: Our support of potato growers and pest management professionals continues to involve personal farm visits, speaking at extension/grower conferences, and reporting to cooperating grower associations. We presented talks and posters with the Wisconsin Potato and Vegetable Growers about our research at their annual Extension Grower Education Conference in February 2019. • Research: We continue to engage with the community of researchers investigating insect pest evolution, and have communicated research findings through regular meetings with collaborators in Vermont and Maryland, and through two presentations at the 2019 Entomological Society of America annual meeting. We published two papers and submitted two additional papers. • Outreach: This research has facilitated the training and engagement of four UW undergraduates and two high school students. In 2019, we supported a Agricultural Research Station intern through this project. We continue seeking outlets for communicating the importance of entomology and insect pest evolution. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has so far provided training opportunities for four undergraduates, two high school students, and training and professional development opportunities for two PhD candidates, Zach Cohen and Yi-Ming Weng. One undergraduate, Mary Magnuson, was supported in this project as a Agricultural Research Station intern in 2019. Zach Cohen,a PhD candidate, submitted his first publication relating to this project. Yi-Ming Weng became a PhD candidate and is working towards his first publication. How have the results been disseminated to communities of interest?We communicated with the Wisconsin Potato and Vegetable Growers about our research at their annual Extension Grower Education Conference in February 2019 in both a talk and poster. Graduate student Zach Cohen presented the results of his research at the 2019 Arthropod Genomics Symposium in Kansas and the 2019 Entomology Society of America Annual meeting in St. Louis. We published two manuscripts, have two in review, and have one more in preparation for peer reviewed publication. What do you plan to do during the next reporting period to accomplish the goals?Objective 1) We will finalize QTL analyses and integrate those into a publication with a new genome assembly for Colorado potato beetle. Objective 2) We will publish our analysis of whole genome resequencing data and the role of standing genetic variation in regional adaptation to insecticides. We will begin analysis of genome sequencing data from museum specimens to complete a temporal analysis of the role of standing genetic variation on insecticide resistance evolution. Results from the efforts will be presented at the Wisconsin Potato and Vegetable Growers Extension Grower Education Conference in February 2020, and the International Congress of Entomology meeting in Finland in 2020.

Impacts
What was accomplished under these goals? Objective 1) We performed an initial data collection of 2 families resulting from crosses of resistant and susceptible populations of Wisconsin Colorado potato beetles. Phenotyping was conducted, and genomic data were collected. In 2019, we developed a new genome assembly and have constructed a linkage map. We are in the process of conducting the QTL study. We also integrated existing transcriptomic data and genome scan data from population resequencing efforts. Two papers have recently been submitted and are in peer-review. We identified metabolic detoxifocation networks (CYP, GST, and ABC transporters) as playing a primary role in resistance. Objective 2) We completed analyses of whole genome resequencing data from across the range of Colorado potato beetles, identifying signatures of standing genetic variation contributing to convergent patterns of resistance in different geographical regions of the USA. Two papers were published in 2019, and one additional manuscript is currently in preparation. We also collected genomic DNA from >250 museum specimens, representing populations from the Midwest and Eastern US, and covering a period of >100 years (including prior to industrial pesticide use). We have sequenced these samples but have not yet analyzed the data. We quantified change in the amount of potato, Solanum tuberosum L., land cover since 1850 using county-level agricultural census reports, obtained allele frequency data from 7,408 single-nucleotide polymorphism loci, and compared effects of historic and contemporary landscape connectivity on genetic differentiation of Colorado potato beetle, Leptinotarsa decemlineata Say, in two agricultural landscapes in the United States. We found that potato land cover peaked in Wisconsin in the early 1900s, followed by rapid decline and spatial concentration, whereas it increased in amount and extent in the Columbia Basin of Oregon and Washington beginning in the 1960s. In both landscapes, we found small effect sizes of landscape resistance on genetic differentiation, but a 20× to 1,000× larger effect of contemporary relative to historic landscape resistances. Demographic analyses suggest population size trajectories were largely consistent among regions and therefore are not likely to have differentially impacted the observed patterns of population structure in each region. Weak landscape genetic associations might instead be related to the coarse resolution of our historical land cover data. Despite rapid changes in agricultural landscapes over the last two centuries, genetic differentiation among L.decemlineata populations appears to reflect ongoing landscape change. The historical landscape genetic framework employed in this study is broadly applicable to other agricultural pests and might reveal general responses of pests to agricultural land-use change.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Crossley, M. S., Rondon, S. I., & Schoville, S. D. (2019). Patterns of genetic differentiation in Colorado potato beetle correlate with contemporary, not historic, potato land cover. Evolutionary applications, 12(4), 804-814.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Crossley, M. S., Rondon, S. I., & Schoville, S. D. (2019). Effects of contemporary agricultural land cover on Colorado potato beetle genetic differentiation in the Columbia Basin and Central Sands. Ecology and evolution, 9(16), 9385-9394.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Hawthorne, D. J., Crossley, M. S., Schoville, S. D., Steinhauer, N., & Dively, G. P. (2019). Regional differences in gene regulation may underlie patterns of sensitivity to novel insecticides in Colorado potato beetle. bioRxiv, 794446.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Cohen, Z. P., Brevik, K., Chen, Y. H., Hawthorne, D. J., Weibel, B. D., & Schoville, S. D. (2019). Elevated rates of positive selection drive the evolution of pestiferousness in the Colorado potato beetle (Leptinotarsa decemlineata, Say). bioRxiv, 870543.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:1) US potato growers and pest management professionals, 2) scientists investigating insect pest evolution, and 3) the Madison community. • Extension: Our support of potato growers and pest management professionals continues to involve personal farm visits, speaking at extension/grower conferences, and reporting to cooperating grower associations. We communicated with the Wisconsin Potato and Vegetable Growers about our research at their annual Research Conference in August 2018. • Research: We continue to engage with the community of researchers investigating insect pest evolution, and have communicated research findings through regular meetings with collaborators in Vermont and Maryland, and through the Entomological Society of America annual meeting. • Outreach: This research has facilitated the training and engagement of four UW undergraduates and two high school students. We continue seeking outlets for communicating the importance of entomology and insect pest evolution. Changes/Problems:Objective 1) We performed an initial data collection of 2 families resulting from crosses of resistant and susceptible populations of Wisconsin Colorado potato beetles. Our initial efforts to identify causative loci produced insignificant results. At present, we are working on improving the genome assembly before continuing to examine other families from our QTL crosses, and we are exploring options to conduct whole genome sequencing (instead of reduced representation sequencing). What opportunities for training and professional development has the project provided?The project has so far provided training opportunities for four undergraduates, two high school students, and training and professional development opportunities for one PhD candidate, Zach Cohen. Zach has advanced to PhD candidacy and has nearly completed his first publication relating to this project. He presented research to the Entomology Society of America in November 2018. How have the results been disseminated to communities of interest?We communicated with the Wisconsin Potato and Vegetable Growers about our research at their annual Research Conference in August 2018. Graduate student Zach Cohen presented the results of his research at the Entomological Society of America annual meeting in November, 2019, in Vancouver, Canada. We also currently have two manuscripts in preparation for peer reviewed publication, and several projects nearing the writing stage. What do you plan to do during the next reporting period to accomplish the goals?Objective 1) We will conduct network analyses of the combined genomic and transcriptomic data, to identify novel metabolic pathways underlying insecticide resistance. We will also extend our effort to map causative loci, by sequencing additional families from crosses. Objective 2) We will publish our analysis of whole genome resequencing data and the role of standing genetic variation in regional adaptation to insecticides. We will conduct genome sequencing of museum specimens to complete a temporal analysis of the role of standing genetic variation on insectide resistance evolution. Results from the efforts will be presented at the Entomology Society of America North Central branch meeting in 2020, and the International Congress of Entomology meeting in Finland in 2020. Additionally, results will be shared with the Wisconsin Potato and Vegetable Growers in February and in August 2019.

Impacts
What was accomplished under these goals? Objective 1) We performed an initial data collection of 2 families resulting from crosses of resistant and susceptible populations of Wisconsin Colorado potato beetles. Phenotyping was conducted, and genomic data were collected. We also integrated existing transcriptomic data and genome scan data from population resequencing efforts. Some initial examination has identified metabolic detoxification networks (CYP, GST, and ABC transporters) as playing a primary role in resistance. Objective 2) We completed analyses of whole genome resequencing data from across the range of Colorado potato beetles, identifying signatures of standing genetic variation contributing to convergent patterns of resistance in different geographical regions of the USA. We also collected genomic DNA from >250 museum specimens, representing populations from the Midwest and Eastern US, and covering a period of >100 years (including prior to industrial pesticide use). We are in the process of sequencing these samples.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Clements, J., S. Schoville, A. Clements, D. Amezian, T. Davis, B. Sanchez-Sedillo, C. Bradfield, A.S. Huseth, and R.L. Groves. 2018. Agricultural fungicides inadvertently influence the fitness of Colorado potato beetles, Leptinotarsa decemlineata, and their susceptibility to insecticides. Scientific Reports 8: 13282.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Pélissié, B., M. Crossley, Z. Cohen, and S.D. Schoville. 2018. Population genomics provide insight on mechanisms of rapid evolution in insect pests. Current Opinion in Insect Science 26: 8-16.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Brevik, K., S.D. Schoville, D. Mota-Sanchez, and Y.H. Chen. 2018. Pesticide durability and the evolution of resistance: A novel application of survival analysis. Pest Management Science 74: 1953⿿1963.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Crossley, M., S.I. Rondon, and S.D. Schoville. 2019. Patterns of genetic differentiation in Colorado potato beetle correlate with contemporary, not historic, potato land cover. Evolutionary Applications.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Schoville, S.D., et al. 2018. A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Science Reports 8: 1931.