MONARCH BUTTERFLY CONSERVATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013527
• Project No.: NEB-28-116
• Multistate No.: NC-1205
• Project Start Date: Oct 1, 2017
• Project End Date: Sep 30, 2022

Project Director
Velez Arango, AN.

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
Entomology

Non Technical Summary
The decline of overwintering monarch butterflies over the last ten yearshas been attributed to the loss of overwintering habitat in Mexico and the loss of seasonal milkweed plants required for larval survival in the U.S. The decline of monarch butterfly populations instigated a petition in 2014 by the U.S. Fish and Wildlife Service, which lists these pollinator insects as a threatened species under the Endangered Species Act. A status review decision is scheduled for 2019. Monarch butterflies found east of the Rocky Mountains have multiple summer generations, with the final generation migrating to an overwintering location in Mexico. It is estimated that half of the monarch butterflies that overwinter in Mexico originate in the Midwest U.S.A primary factor affecting monarch larvae is the loss of larval habitat due to an increase in acreage for crop production. The crop acreage in Nebraska has increased from 8.5 to 9.4 million acres in the past 10 years, and natural areas are often in proximity to corn and soybean row crops. A survey of 560 locations in Nebraska found 9,320 milkweed plants with a total of 14 and 8 monarch larvae in 2015 and 2016, respectively. Fluctuating climate and agricultural practices can affect the reproduction and longevity of monarch populations; however, there is a gap in knowledge regarding the impact of these stressors on the survivorship of monarch larvae. This project aims to identify stressors that affect monarch survival in the Midwest U.S. by continuing surveying monarch abundance and milkweed habitats.Furthermore, we will evaluate the effects of starvation and exposure to pesticides conventionally used across agricultural habitats of Nebraska on monarch larvae. Monarch larvae exposed to these stressors will be compared to controls to determine effects on lipid metabolism and samples will be collected to perform RNA-sequencing to identify stressor-specific biomarkers that could be used to monitor monarch population health. In the long term, this information will contribute to the identification and implementation of mitigation strategies to protect an important pollinator in Nebraska.?

Animal Health Component

0%

Research Effort Categories

Basic

90%

Applied

0%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3095	1130	100%

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

Subject Of Investigation
3095 - Other non-bee pollinators, general;

Field Of Science
1130 - Entomology and acarology;

Keywords

monarch butterfly

conservation

environmental stressors

fitness effects

milkweed

pesticides

lipid metabolism

starvation

survival

biomarkers

Goals / Objectives
DETERMINE OPTIMAL BREEDING HABITAT PATCH CHARACTERISTICS AND LANDSCAPE SPATIAL ARRANGEMENTS TO MAINTAIN AND PROMOTE MONARCH POPULATION VIABILITY AND DEVELOP A MODEL-BASED DECISION SUPPORT SYSTEM TO GUIDE CONSERVATION RECOMMENDATIONS ACROSS THE EASTERN MONARCH BUTTERFLY RANGE. ESTABLISH SURVEY AND SAMPLING PROTOCOLS THAT CAN BE APPLIED REGION-WIDE TO EVALUATE BREEDING HABITAT QUALITY, MONARCH HABITAT UTILIZATION, AND LIFE STAGE ASSESSMENT FOR A REGION-WIDE MONARCH LIFE CYCLE BASELINE.

Project Methods
MethodsObjective 1: Monarch habitat patches surveyHabitat surveys will be conducted during week-long sampling trips on eight separate dates from May through August over four years. Milkweed plants found growing adjacent to and distal from irrigated and non-irrigated agricultural fields throughout the state of Nebraska will be sampled. Plants to be surveyed will be selected based on distance from the edge of fieldsbut will be approximately 0-5, 5-25, and more than 25 meters from the nearest field borders. At least 12 sites will be sampled each day of the survey. The goal is to have even sampling in agriculturally intensive, and in areas where crops production is reduced or absent.For each plant observed, monarch caterpillars and other insects will be counted. If identification is uncertain, specimens will be collected and taken to the lab for minimal family, and ideally species identification. Ten to 15 plants will be sampled at each location. At each location, milkweed species sampled, the diversity of other plant species in the area and GPS coordinates will be recorded.We have two years of preliminary data from 2015-2016. After four more years of sampling, we will begin to look for correlations of monarch populations, and the abundance of other insects on milkweed with proximity to agricultural fields. Analysis of land use patterns at each sampling location will also be determined using the USDA's National Agriculture Statistics Service Cropland Data Layer, CropScape.Objective 2: Fitness effects of common stressorsA colony of monarch butterflies will be established at the University of Nebraska-Lincoln to perform the fitness and lipid metabolism experiments. Researchers from Iowa State University that are part of the NC_temp 1205 Committee will provide eggs to establish the colony and provide expertise on rearing strategies.We will evaluate the fitness effects of common stressors found in the agricultural landscapes of Nebraska including the insecticides imidacloprid and bifenthrin, the herbicide glyphosate, and starvation on monarch larvae. We will measure different fitness parameters including larval development, the rateof pupation, and the fertility and fecundity of adults exposed as larvae. These parameters will allow us to calculate population growth,comparedto unexposed insects, and determine the effects of these stressors on population growth.Objective 3: Effect of pesticide exposures on monarch larvae lipid metabolism and functionLipids are of vital importance to the monarch butterfly as substrates for embryogenesis,metamorphosis and flight. The function and metabolism of lipidshavebeen well studied in recent years; however, it is unclear how pesticide exposures, and other stressors, might affect the total lipids and lipid fractions in the monarch butterfly and, in turn, the health of this pollinator. Carboxylesterases are a superfamily of serine hydrolases that hydrolyze ester bonds, amide bonds, etc. of both endogenous (lipids) and exogenous substrates (pesticides). There is a gap in knowledge regarding the physiological functions of carboxylesterases, their interactions with pesticides and lipids, and the health implications of these interactions in the monarch butterfly. Here, we will examine the (i) carboxylesterase enzyme activities and gene expression, (ii) total lipids and lipid fractions (free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, cholesterol, etc.), and (iii) selective inhibition of carboxylesterases on total lipids and lipid fractions of pesticide- and starvation-stressed monarch butterflies.Objective 4: RNA-sequencing of pesticide- and starvation-stressed monarch larvaeWe will perform RNA-sequencing of larvae exposed to the stressors indicated in objective 2. RNA-sequencing data of larvae exposed to stressors will be compared to control larvae to identify genes that are differentially expressed. We will mainly focus on genes involved with toxins metabolism, lipid sequestration, and other genes reported to be involved in stress responses. The identification of these genes will allow us to better understand stress responses in monarch larvae and will provide us with a baseline for future studies. Additionally, this information could allow us to identify marker genes that could potentially be tested in field populations.Milestones and TimelineThe proposed research is expected to take place between October2017 to September2021. A timetable for completing each objective is outlined below.Objective 1: Monarch habitat patches SurveyAim 1. Summer surveys: Summer 2018 -2020Aim 2. Correlation analysis: Spring 2020 - Fall 2021Objective 2: Fitness effects of common stressorsAim 1. Establish butterflies' colony: Fall 2017 - Spring 2018Aim 2. Identify pesticides sublethal concentrations: Summer 2018 - Spring 2019Aim 3. Perform fitness evaluations: Fall 2018 - Fall 2020Objective 3: Effect of pesticide exposures on monarch larvae lipid metabolism and functionAim 1. Carboxylesterase enzyme activity and gene expression: Fall 2017 - Spring 2019Aim 2. Total lipids and lipid fractions: Fall 2018 - Fall 2020Aim 3. Selective inhibition of carboxylesterases on total lipids and lipid fractions of pesticide- and starvation-stressed monarch butterflies: Sring 2019 - Fall 2020Objective 4: RNA-sequencing of pesticide- and starvation-stressed monarch larvaeAim 1. Collect samples: Fall 2018 - Spring 2020Aim 2. Bioinformatics analysis: Spring 2020 - Summer 2021Propagate and report the results obtainedAim 1. Write papers and reports: Spring 2020 - Fall 2021Aim 2. Write and submit USDA AFRI Grant for New Frontiers in Pollinator Health: Summer 2021

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:The target audience reached during this reporting period included academic, government, and industry scientists, general public, and undergraduate students from Insect Science, Biological Sciences, Environmental Studies, and CASNR Integrated Science. Additionally, Co-PD Anderson hosted one CASNR CUSP Scholar from Rwanda. Two undergraduate students were selected to work on this project during the summer of 2021 as part of the Research and Extension Experiences for Undergraduates (REEU) on Beneficial Arthropods. The students conducted research with monarch butterflies and pesticides and one of them assisted with colony maintenance. Changes/Problems:The following changes have been performed: Change in the initial Objective 3:Measure the effect of pesticide exposure on monarch larvae lipid metabolism and function was adjusted to fit the Ph.D. student research interests. It was changed to measure the impact of pesticide exposure and other chemical stressors (i.e., different cardenolide levels, KCL) on monarch larvae survival. This objective still evaluated the effects of pesticide exposure and tested the impact of other compounds such as crease in cardenolides and KCl (a commonly used fertilizer in Nebraska Agriculture) on monarch larvae survival. Furthermore, instead of evaluating the effects of pesticide exposure on lipid metabolism and function, the student assessed the activity and gene expression of detoxification enzymes, which is a more immediate need to understand pesticide's effect on monarchs better to improve conservation strategies. Change in the initial Objective 4: Perform RNA-sequencing of monarch larvae exposed to common agricultural stressors to characterize changes in gene expression and potentially identify stressor-specific biomarkers. In 2020, we started a collaboration with Iowa State University to collect samples for the RNA-sequencing proposed in this objective. However, samples were collected at different time points, and they will not be useful to obtain adequate RNA-sequencing data. The student that collected the samples graduated. However, we observed high variability in gene expression between the samples evaluated in objective 3 with RT-qPCR. These results evidenced the need to increase the number of biological replicates for RNA sequencing to five insects per treatment, limiting the number of treatments that could be evaluated given the cost of RNA sequencing. It was also determined that a baseline transcriptome for the different life stages and types of diets used in objectives 2 and 3 should be performed first before evaluating the effects of pesticides on global gene expression levels. For that reason, it was decided to perform a transcriptome for second, third, and fifth instar caterpillars feeding on artificial diet and tropical milkweed lead diet. The generated transcriptomes will be published and used in future research to study the effects of pesticides on gene expression. Data for this objective is currently being analyzed. The Nebraska Monarch Conservation summit was canceled, given the continued restrictions with COVID-19. What opportunities for training and professional development has the project provided?The project has provided opportunities for research training of the Ph.D. and M.S. graduate students and undergraduate students. Including: Annie Krueger, Ph.D. UNL Department of Entomology Matthew Greiner, M.S. student at UNL Department of Entomology Samantha Kiever, B.S. student at UNL Department of Entomology Edith Ikuze, BS, Integrated Science, UNL CASNR CUSP Fellow Bennet Grappone, atB.S. student at UNL Department of Entomology Abigail Berrios-Starbird, visiting USDA REEU research assistant, B.S. student at St. Edwards University, Austin, TX Breighton Reed, visiting USDA REEU research assistant, B.S. student at St Petersburg College, FL Furthermore, this project has fostered collaborations with researchers at the University of Nebraska-Lincoln (UNL) and other institutions. Including: Drs. Steve Levine (Bayer CropScience) and Thomas Steeger (US EPA) were mentors for a Foundation for Food and Agricultural Research Professional Development Fellowship awarded to AK Dr. Daniel Swale at Louisiana State University Department of Entomology is assisting with measuring the effects of neuronal function in monarchs after direct exposure to cardenolides and pyrethroids Dr. Kathryn Hanford at UNL Department of Statistics is assisting with statistics of monarch exposures to cardenolides and pyrethroids Dr. Kristi Montooth and Ph.D. Candidate Cole Julick at UNL School of Biological Sciences to quantify temperature effects onmonarch larval growth and feeding rates Dr. Steve Bradbury, Dr. Richard Helmich, and Ph.D. student Niranjana Krishnan at Iowa State University, Department of Entomology. Monarch colony rearing Dr. Brigitte Tenhumberg and Ph.D. student Chih-Chung Lee at UNL School of Biological Sciences to quantify cardenolide levels in milkweeds Dr. Christopher Brown, Bayer CropScience, collaboration to sustain monarch colony at UNL after Hatch Project ends How have the results been disseminated to communities of interest?The results have been disseminated by presenting posters at multiple scientific societies meetings, including American Chemical Society (ACS), Society of Environmental Toxicology and Chemistry (SETAC), and Entomological Society of America (EntSoc). The result have also been disseminated in the PD classes Toxins in the Environment and Insecticide Toxicology. What do you plan to do during the next reporting period to accomplish the goals?Under each objective, we plan to perform the following activities to accomplish the goals. Objective 1. Surveys for monarch butterfly larvae and other endemic arthropods found on milkweed plants will be conducted across Nebraska in 2022, and data will be analyzed. Objective 2. Publish comparison on monarch development between artificial diet and tropical milkweed. Complete exposures of mixtures between the insecticides bifenthrin, chlorpyrifos, and thiamethoxam, and the fungicide propiconazole and prepare publication. Graduation of M.S. student Mathew Greiner. Objective 3. Revision on manuscript submitted. Objective 4. Finish analyzing RNA-sequencing data and prepare publication.

Impacts
What was accomplished under these goals? This project aims to identify stressors that affect monarch butterflyDanaus plexippussurvival in the Midwest U.S. We have continued to survey and demonstrate a decline of monarchs in Nebraska. Insecticide exposure has been identified as a contributing stressor to the decline in the monarch butterfly population. The research performed evaluated lethal and sublethal effects of commonly used insecticides and fungicides in Nebraska at field-realistic concentrations. The information obtained in this project will contribute to identifying and implementing mitigation strategies to protect monarch butterflies and the decision to list monarchs as Endangered Species in 2024. Goal 1, Objectives 2, 3, and 4 Objective 2. Measure fitness costs of common stressors experienced by monarch larvae in agricultural environments. The final replication to determine the difference between leaf-fed and artificial diets. Insects were followed until adult emergence, observation of daily instar change, mortality, and pupal weight was performed. Artificial diet increased the developmental time but showed a higher pupal weight. Overall survival was similar, adults were healthy, and rearing in the diet has been successful. Oral toxicity of the insecticides bifenthrin, chlorpyrifos, and thiamethoxam at field-realistic concentrations was evaluated in third instar monarchs in three replications. Third instar larvae were exposed to each insecticide for 96-hours using acetone and water controls. Exposures were based on modeled pesticide drift with high spray label rates of formulated products at 0, 5, and 10 m away from application fields. Lethality and observations on behavior were recorded until adult emergence. Mixture toxicity bioassays with the insecticides bifenthrin, chlorpyrifos, and thiamethoxam with the fungicide propiconazole were performed using the same levels described in the previous experiment. To date, the only effect observed is that propiconazole reduces toxicity to chlorpyrifos. Objective 3.Measure the effect of pesticide exposure on monarch larvae lipid metabolism and function. Milkweed species affect detoxification enzyme activity and expression in monarch caterpillars. We investigated the effects of cardenolide composition on pyrethroid sensitivity and detoxification enzymes of monarch caterpillars feeding on a high cardenolide milkweed, tropical milkweed, or a low cardenolide, swamp milkweed. Caterpillars fed on each plant through the fifth-instar stage and were topically treated with bifenthrin after reaching the final-instar. Detoxification enzyme activity and gene expression were quantified for esterase, glutathione S-transferase, and cytochrome P450 enzymes and transcripts encoding a selected set of these enzymes. Bifenthrin exposure and different milweed species affected both enzyme activity and gene expression. These results suggest that feeding on different milkweed species can affect detoxification mechanisms with which monarch caterpillars rely on to cope with chemical stressors. This study was submitted to the journal Pesticide Biochemistry & Physiology. Cardenolide, potassium, and pyrethroid insecticide combinations reduce growth and survival of monarch butterfly caterpillars. This study investigated the growth, survival, and development of caterpillars exposed to the cardenolide ouabain, bifenthrin, and potassium chloride (KCl) alone and in combination. Caterpillar weight, diet consumption, frass, and survival were recorded for the duration of the experiments. It was observed that ouabain increased body weight and diet consumption, whereas KCl decreased body weight and diet consumption. Caterpillars feeding on KCl and treated with bifenthrin consumed less diet compared to individuals provided untreated diet. However, there was no effect on survival or body weight. Combinations of KCl + ouabain did not significantly affect caterpillar survival or body weight following treatment with bifenthrin. At the concentrations tested, there were no effects observed for bifenthrin sensitivity with increasing cardenolide or KCl concentrations. Further studies are warranted to understand how milkweed-specific cardenolides and agrochemical inputs can affect monarch caterpillar physiology near agricultural landscapes. This study was published in the Journal of Economic Entomology. Pyrethroid exposure reduces growth and development of monarch butterfly caterpillars. This study examined the 72-h toxicity of two pyrethroid insecticides, bifenthrin and β-cyfluthrin, and their effects on growth and diet consumption. The toxicity of bifenthrin to caterpillars was lower than β-cyfluthrin after 72 h. Survival was the most sensitive endpoint for bifenthrin, but diet consumption and caterpillar growth were significantly reduced at sublethal levels of β-cyfluthrin. Using AgDRIFT spray drift assessment, the aerial application of bifenthrin or β-cyfluthrin is predicted to pose the greatest risk to fifth-instar caterpillars, with lethal insecticide deposition up to 28 m for bifenthrin and up to 23 m for β-cyfluthrin from treated edges of fields. Low boom ground applications are predicted to reduce distances of lethal insecticide exposure to 2 m from the treated field edge for bifenthrin and β-cyfluthrin. Growth and survival of fifth-instar caterpillars developing within the margins of a treated field may be significantly impacted following foliar applications of bifenthrin or β-cyfluthrin. These findings provide evidence that pyrethroid insecticides commonly used for soybean pest control are a potential risk to monarch caterpillars in agricultural landscapes. This study was published in the Journal of Insect Science. Objective 4.Perform RNA-sequencing of monarch larvae exposed to common agricultural stressors to characterize changes in gene expression and potentially identify stressor-specific biomarkers. Given that we do not have a baseline transcriptome for monarchs and our experiments were performed at different life stages and artificial and leaf diets, we decided to first determine the gene expression effects of the different diets on second, third, and fifth instar larvae. Samples were collected, processed and sent for RNA sequencing. Results are currently being analyzed. This transcriptomic data will serve as a baseline to evaluate the effect of pesticides on gene expression in the future. Goal 2, Objective 1 Objective 1. Characterize habitat patches and landscape spatial arrangements in Nebraska and correlate with monarch larval abundance. Surveys for monarch butterfly larvae and other endemic arthropods found on milkweed plants were conducted across Nebraska. A total of 7,234, 5,563, 4,662, 2,750, and 7,257 milkweed (Asclepias spp.) plants were surveyed in 2017, 2018, 2019, 2020, and 2021, respectively. Patches of milkweed, were selected from variable habitats along roads, adjacent to agricultural areas, and in state parks or federal lands. Sites sampled totaled 707 in 2017, 447 in 2018, 290 in 2019, 203 in 2020 and 418 in 2021. All plants, or a subsample of plants in a patch were inspected and arthropods on plants were counted and their numbers recorded. Plants or crops in land adjacent to sampling locations were also noted. A total of 15, 79, 90, 18, and 79 monarch butterfly larvae were observed in 2017, 2018, 2019, 2020, and 2021, respectively. Monarch caterpillars were observed feeding on common, showy, sand, green comet, swamp, whorled, and spider milkweed species. Larvae were most commonly encountered on common (143 caterpillars/17,636 stems = 0.81% utilization) and sand (52 caterpillars/4,181 stems = 1.2% utilization) milkweed species. A correlation analysis between monarch caterpillar abundance, and sampling location as well as landscape ecology, land use patterns, and the abundance of flowering plants will be conducted.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Krueger, A. J., Rault, L. C., Velez, A. M., Weissling, T. D., and Anderson, T. D. 2020. Three-way interaction of pyrethroid insecticide, cardiac glycoside, and potassium fertilizer to monarch butterfly caterpillars. Oral presentation by AJK at Society of Environmental Toxicology & Chemistry North America 41st Annual Meeting, November 15-19, 2020 (Virtual).
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Greiner, M. A., Krueger, A. Spencer, T., Anderson, T., Smart, A., Weissling, T. and V�lez, A.M. 2020. Impact of Fungicide-Insecticide Interactions on Monarch (Danaus plexippus L.) Caterpillars. Oral presentation by MG at Entomological Society of America Annual Meeting, November 11-25, 2020 (Virtual).
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Greiner, M. A., Krueger, A. J., Anderson, T. D., Smart, A., Weissling, T., and V�lez A. M. 2021. Impact of fungicide-insecticide interactions on monarch (L. Danaus plexippus) caterpillars. Oral presentation by MG at Society of Environmental Toxicology and Chemistry Ozark-Prairie and Mid-South Regional Meeting, May 12, 2021. First place in the student presentation competition.
• Type: Conference Papers and Presentations Status: Under Review Year Published: 2021 Citation: (Poster) Berrios-Starbird, A. K., and Anderson, T. D. 2021. Effects of chlorpyrifos-oxon and piperonyl butoxide on acetylcholinesterase activity in monarch butterflies. Poster presentation by AKB at the University of Nebraska Summer Research Fair, July, 2021.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Krueger, A. J., Hanford, K. J., Weissling, T., Velez, A. M., and Anderson, T. D. 2021. Pyrethroid exposure reduces growth and development of monarch butterfly (Lepidoptera: Nymphalidae) caterpillars. Journal of Insect Science. Volume 21, Issue 2, Pages 1-8 (Accepted 13 December 2020, Published 4 March 2021).
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Krueger A. J. Robinson E. A., Weissling T. J., V�lez A. M., and T. D. Anderson. 2021. Cardenolide, Potassium, and Pyrethroid Insecticide Combinations Reduce Growth and Survival in Monarch Caterpillars (Lepidoptera:Nymphalidae). Journal of Economic Entomology. Volume 114, Issue 6, Pages 23702380 (Accepted: 17 August 2021, Published: 17 September 2021).
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Krueger, A. J., Rault, L. C., Robinson, E. A., Weissling, T. J., Velez, A. M., and Anderson, T. D. 2022. Milkweed species affect detoxification enzyme activity and expression in monarch caterpillars. Pesticide Biochemistry and Physiology (In review).
• Type: Theses/Dissertations Status: Published Year Published: 2021 Citation: Krueger, A. J. 2021. Toxicology of Chemical Stress to Monarch Butterflies (Danaus plexippus L.). Advisor: Anderson, T.D. Published: August 2021. University of Nebraska-Lincoln, Department of Entomology.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Krueger, A. J., Velez, A. M., Rault, L. C., Weissling, T., and Anderson, T. D. 2020. Host plant effects on insecticide detoxification gene expression in monarch butterflies. Oral presentation by AJK at Entomological Society of America Meeting, November 11-25, 2020 (Virtual).
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: (Poster) Reed, B. S., Greiner, M. A., Kruger, A. J. and V�lez A. M. 2021 Effects of Tropical Milkweed (Asclepias curassavica L.) in the Artificial Diet of Monarch Butterfly (Danaus plexippus L.). Poster presentation by RBS at the University of Nebraska Summer Research Fair, July, 2021.
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Greiner, M.A., Krueger, A., Anderson, T., Smart, A., Weissling, T., and V�lez A. 2021. Impact of Multiple Pesticide Exposures on Monarch Caterpillars in Agricultural Systems. Oral presentation by MG at University of Nebraska-Lincoln CASNRs Elevator Speech Contest, October, 2021.
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Greiner, M.A., Krueger, A., Anderson, T., Smart, A., Weissling, T.,and V�lez A. 2021. Assessing Monarch Caterpillar (Danaus plexippus L.) Survival from Exposure to Fungicide-Insecticide Combinations. Oral presentation by MG at Society of Environmental Toxicology and Chemistry Annual Meeting, November 14-18, 2021 (Virtual).

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

Outputs
Target Audience:The target audience reached during this reporting period included academic, government, and industry scientists, general public, and undergraduate students from Insect Science, Biological Sciences, Environmental Studies, and CASNR Integrated Science majors. Additionally, Co-PDAnderson hosted one CASNR CUSP Scholar from Rwanda. The students conducted research with monarch butterflies and pesticides and assisted with colony maintenance. Two undergraduate students were selected to work on this project during the summer of 2020 as part of the Research and Extension Experiences for Undergraduates (REEU) on Beneficial Arthropods. However, due to Covid-19, all REEU experiences at UNL were canceled. Co-PDWeissling and graduate student Annie Krueger worked with representatives from Nebraska Department of Transportation, Nebraska Game and Parks, Pheasants Forever, and Xerces Society to organize a state-wide pollinator conservation meeting for April 2020. The meeting's goal was to connect state conservation efforts and encourage collaboration and data sharing through breakout groups. Special Projects funding was awarded from UNL to host the summit; however, due to Covid-19, summit planning was placed on hold for the remainder of 2020. We will aim to host the summit in 2021. Changes/Problems:COVID-19 slowed the progress of the experiments planned for 2020, as the University of Nebraska-Lincoln restricted students' access for a month. After re-entrance, only one individual at a time was allowed in the laboratory. Furthermore, due to COVID-19, we were not able to host the two USDA NIFA REEU undergraduate research fellow we had scheduled to work on monarch butterflies. We started a collaboration with Iowa State University to collect samples for the RNA-sequencing proposed in Objective 4. However, samples were collected at different time points, and they will not be useful to obtain adequate RNA-sequencing data. Samples for RNA-sequencing will be collected in the Spring of 2021. We will also aim tohost the Nebraska Monarch Conservationsummit in 2021. What opportunities for training and professional development has the project provided?The project has provided opportunities for research training of the Ph.D. and M.S. graduate students and undergraduate students. Furthermore, this project has fostered collaborations with researchers at the University of Nebraska-Lincoln (UNL) and other institutions. These collaborations include: Annie Krueger, Ph.D. candidate at UNL Department of Entomology, Foundation for Food and Agricultural Research Professional Development Fellow. Matthew Greiner, M.S. student at UNL Department of Entomology. Drs. Steve Levine (Bayer CropScience) and Thomas Steeger (US EPA) are industry and government mentors, respectively, for a Foundation for Food and Agricultural Research Professional Development Fellowship awarded to Annie Krueger.Dr. Daniel Swale at Louisiana State University Department of Entomology is assisting with measuring the effects of neuronal function in monarchs after direct exposure to cardenolides and pyrethroids. Dr. Autumn Smart at UNL Department of Entomology is assisting with biostatistics and modeling of monarch exposures to cardenolides and pyrethroids. Dr. Kathryn Hanford at UNL Department of Statistics is assisting with statistics of monarch exposures to cardenolides and pyrethroids. Dr. Kristi Montooth and Ph.D. student Cole Julick at UNL School of Biological Sciences to quantify temperature effects on monarch larval growth and feeding rates. Dr. Steve Bradbury, Dr. Richard Helmich, and Ph.D. student Niranjana Krishnan at Iowa State University, Department of Entomology. Monarch colony rearing and RNA sequencing. Dr. Brigitte Tenhumberg and Ph.D. student Chih-Chung Lee at UNL School of Biological Sciences to quantify cardenolide levels in milkweeds. Dr. Christopher Brown, Bayer CropScience, collaboration to sustain monarch colony at UNL after Hatch Project end date. How have the results been disseminated to communities of interest?The results have been disseminated by presenting posters at multiple scientific societies meetings, including American Chemical Society (ACS), Society of Environmental Toxicology and Chemistry (CETAC), and Entomological Society of America (EntSoc). The result have also been disseminated in the PD classes Toxins in the Environment and Insecticide Toxicology, and the Nebraska Games and Parks10thLegacy Conference.Co-PDWeissling and graduate student Annie Krueger worked with representatives from Nebraska Department of Transportation, Nebraska Game and Parks, Pheasants Forever, and Xerces Society to organize a state-wide pollinator conservation meeting for April 2020. The meeting's goal was to connect state conservation efforts and encourage collaboration and data sharing through breakout groups. Special Projects funding was awarded from UNL to host the summit; however, due to Covid-19, summit planning was placed on hold for the remainder of 2020. We will aim to host the summit in 2021. What do you plan to do during the next reporting period to accomplish the goals?Under each objective, we plan to perform the following activities to accomplish the goals. Objective 1.Surveys for monarch butterfly larvae and other endemic arthropods found on milkweed plants will be conducted across Nebraska in 2021. Objective 2.Complete replication for comparison on monarch development between artificial diet and tropical milkweed. Complete exposures of mixtures between the insecticidesbifenthrin, chlorpyrifos, and thiamethoxam, and the fungicide propiconazole. Evaluate esterase and P450 levels of larvae exposed to insecticide-fungicide combinations. Objective 3.Examine the effects of swamp, common, and tropical milkweed containing low, medium, and high cardenolide levels, respectively, on pyrethroid insecticide toxicity and detoxification in monarch butterfly caterpillars. Continue determining the effects of pyrethroid and cardenolide exposure on neuronal function in monarch caterpillars. Objective 4.Collect, process and send samples for RNA-sequencing of fifth monarch larvae exposed to thiamethoxam.

Impacts
What was accomplished under these goals? This project aims to identify stressors that affect monarch butterfly Danaus plexippus survival in the Midwest U.S. We have continued to survey and demonstrate a decline of monarchs in Nebraska. Insecticide exposure has been identified as a contributing stressor to the decline in themonarch butterfly population. Monarch toxicity data is currently limited, and available data focuses on lethal endpoints. The research performed evaluated lethal and sublethal effects of commonly used insecticides, and fungicides in Nebraska at field-realistic concentrations derived AgDRIFT® spray drift assessment. The information obtained in this project will contribute to identifying and implementing mitigation strategies to protect monarch butterflies and the decision to list monarchs as an Endangered Species in 2024. Goal 1, objectives 2, 3, and 4 Objective 2.Measure fitness costs of common stressors experienced by monarch larvae in agricultural environments.The monarch colony continues to remain healthy and productive.Two replications were performed to determine the difference in the development between leaf fed and artificial diet. Insects were followed until adult emergence, observation daily instar change, mortality, and pupal weight. Artificial diet increased the developmental time but showed a higher pupal weight. Overallsurvival was similar, and adults were healthy, and rearing in the diet has been successful. Understanding the difference will allow us to make better inferences in future experiments performed with artificial diet. Oral toxicity of the insecticides bifenthrin, chlorpyrifos, and thiamethoxam at field-realistic concentrations was evaluated in third instar monarchs using the artificial diet. Third instar larvae were exposed to each insecticide for 96-hours using acetone and water as the controls. Field Realistic pesticide exposures were based on modeled pesticide deposition in the AgDRIFT® spray drift assessment with high spray label rates of formulated products at 0, 5, and 10 meters (m) away from application fields. Lethality and observations on changes of behavior were recorded. Insects were evaluated until adult emergence. Bifenthrin exposure at estimated concentrations to 0 to 47% mortality and no caterpillars survived to adulthood. Chlorpyrifos exposures only had 66% survival at the highest distance (10 m), and only 35% of these caterpillars exposed survived to adulthood. Thiamethoxam exposure did not cause mortality and any of the evaluated levels, but caterpillars feeding at the highest level (0 m) had only a 60% survival to adulthood. One replication of mixture toxicity bioassays with the insecticides bifenthrin, chlorpyrifos, and thiamethoxam with the fungicide propiconazole was performed using the same levels described in the previous experiment. To date, the only effect that we have observed is that propiconazole reduces toxicity to chlorpyrifos. Objective 3.Measure the effect of pesticide exposure on monarch larvae lipid metabolism and function.This study examined the 72-h toxicity of two pyrethroid insecticides, bifenthrin and β-cyfluthrin, and their effects on growth and diet consumption.The toxicity of bifenthrin to caterpillars was lower than β-cyfluthrin after 72 h. Survival was the most sensitive endpoint for bifenthrin, but diet consumption and caterpillar growth were significantly reduced at sub-lethal levels of β-cyfluthrin. Using AgDRIFT®spray drift assessment, the aerial application of bifenthrin or β-cyfluthrin is predicted to pose the greatest risk to fifth instar caterpillars, with lethal insecticide deposition up to 28 m for bifenthrin and up to 23 m for β-cyfluthrin from treated edges of fields. Low boom ground applications are predicted to reduce distances of lethal insecticide exposure to 2 m from the treated field edge for bifenthrin and β-cyfluthrin.Growth and survival of fifth instar monarch caterpillars developing within the margins of a treated field may be significantly impacted following foliar applications of bifenthrin or β-cyfluthrin. These findings provide evidence that pyrethroid insecticides commonly used for soybean and corn pest control are a potential risk to monarch caterpillars in agricultural landscapes.This study was acceptedforpublication tothe Journal of Insect Science. Monarchs are not only exposed to insecticides but also the insecticidal cardiac glycosides in milkweed. Pharmacological studies have shown potassium to antagonize these compounds. Due to this mechanistic congruence, we are exploring the interaction of pyrethroid insecticide (bifenthrin), cardiac glycoside (ouabain), and potassium fertilizer (potassium chloride) on the growth, survival, and detoxification enzyme activity and expression of monarch caterpillars. This study focuses on the toxicology of bifenthrin, with and without co-exposure of ouabain, to fifth instar monarch caterpillars. A two-way interaction showing bifenthrin sensitivity to be altered with ouabain exposure was further examined as well as a three-way interaction with potassium chloride. Additionally, the growth and diet consumption of monarch caterpillars was significantly reduced with two- and three-way exposure to bifenthrin, ouabain, and potassium chloride. These exposure combinations were also shown to differentially regulate the activity and expression of neurophysiological and detoxification genes in monarch caterpillars, including Na+/K+-ATPase, esterase, cytochrome P450, glutathioneS-transferase, and ABC transporter activity and/or expression levels across treatments.A manuscript of this study is in preparation for submission to the peer-reviewed journal Pesticide Biochemistry and Physiology. Objective 4.Perform RNA-sequencing of monarch larvae exposed to common agricultural stressors to characterize changes in gene expression and potentially identify stressor specific biomarkers.A collaboration with Iowa State University was initiated for this objective as they have observed an effect on pupation of fifth instar larvae exposed to Thiamethoxam. Goal 2, objective 1 Objective 1. Characterize habitat patches and landscape spatial arrangements in Nebraska and correlate with monarch larval abundance.Surveys for monarch butterfly larvae and other endemic arthropods found on milkweed plants were conducted across Nebraska. A total of 7,234, 5,563, 4,662 and 2,750 milkweed (Asclepiasspp.) plants were surveyed in 2017, 2018, 2019, and 2020, respectively. Patches of milkweed (14 different species) included in surveys were selected from variable habitats along roads, adjacent to agricultural areas, and in state parks or federal lands. Sites sampled totaled 707 in 2017, 447 in 2018, 290 in 2019 and 203 in 2020. All plants, or a subsample of plants in a patch were carefully inspected and arthropods on plants were counted and their numbers recorded. GPS coordinates of each sampling location were also recorded. In addition, the diversity of flowering plants and overall plant diversity was estimated at several sampling locations. Plants or crops in land adjacent to sampling locations were also noted. A total of 15, 79, 90, and 18 monarch butterfly larvae were observed in 2017, 2018, 2019, and 2020, respectively. Monarch caterpillars were observed feeding on common, showy, sand, green comet, swamp, and spider milkweed species. Larvae were most commonly encountered on common (109 caterpillars/13336 stems = 0.82% utilization) and sand (37 caterpillars/3531 stems = 1.05% utilization) milkweed. Additional arthropods observed included several species of predators, herbivores, and a variety pollinators. A correlation analysis between monarch caterpillar abundance, and sampling location as well as landscape ecology, land use patterns, and the abundance of flowering plants will be conducted when data collection is concluded. The same analysis will be conducted with beneficial insect and pollinator abundance and diversity.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Krueger, A. J., Hanford, K. J., Weissling, T., Velez, A. M., and Anderson, T. D. 2020. Pyrethroid exposure reduces growth and development of monarch butterfly (Lepidoptera: Nymphalidae) caterpillars. Journal of Insect Science (Accepted 13 December 2020).
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Krueger, A. J., Rault, L. C., Velez, A. M., Weissling, T., and Anderson, T. D. 2020. Host plant effects on insecticide detoxification: Cardenolide and pyrethroid exposure affects gene expression in monarchs. Oral presentation by AJK at the American Chemical Society National Meeting & Exposition, San Francisco, CA, August 16-20 (Virtual).
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Krueger, A. J., Rault, L. C., Velez, A. M., Weissling, T., and Anderson, T. D. 2020. Three-way interaction of pyrethroid insecticide, cardiac glycoside, and potassium fertilizer to monarch butterfly caterpillars. Oral presentation by AJK at Society of Environmental Toxicology & Chemistry North America 41st Annual Meeting, November 15-19 (Virtual).
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Krueger, A. J., Velez, A. M., Rault, L. C., Weissling, T., and Anderson, T. D. 2020. Host plant effects on insecticide detoxification gene expression in monarch butterflies. Oral presentation by AJK at Entomological Society of America Annual Meeting, November 11-25 (Virtual).
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Greiner, M., Krueger, A. Spencer, T., Anderson, T., Smart, A., Weissling, T. and V�lez, A.M. 2020. Impact of Fungicide-Insecticide Interactions on Monarch (Danaus plexippus L.) Caterpillars. Oral presentation by MG at Entomological Society of America Annual Meeting, November 11-25 (Virtual).

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

Outputs
Target Audience:The target audience reached during this reporting period included the general public and University of Nebraska-LIncoln undergraduate students from Insect Science, Biology, Environmental Restoration Science and Environmental Studies majors. Additionally, we hosted one undergraduate student from Lock Haven University during the summer of 2019 as part of the Research and Extension Experiences for Undergraduates (REEU) on Beneficial Arthropods. The student performed research with pesticides and monarchs. Changes/Problems:Experiments to identify sublethal concentrations of pesticides and the collection of samples for RNA-sequencing were delayed due to issues establishing the monarch colony. The colony is now well established, and the rearing method has been standardized. An additional delay to objective 2 and 4 was due to the resignation of a graduate student in the Fall of 2018. A new M.S. student was hired in the summer of 2019. What opportunities for training and professional development has the project provided?The project has provided opportunities for research training of the Ph.D. and M.S. graduate students and undergraduate students. Furthermore, this project has fostered collaborations with researchers at the University of Nebraska-Lincoln (UNL) and other institutions. These collaborations include: Dr. Kristi Montooth and Ph.D. student Cole Julick at UNL School of Biological Sciences to quantify temperature effects on monarch larval growth and feeding rates. Dr. Steve Bradbury, Dr. Richard Helmich, and Ph.D. student Niranjana Krishnan at Iowa State University, Department of Entomology. Comparison of toxicity endpoints across laboratories and development of larval exposure estimates based on consumption and body mass. We are fostering future collaborations with Dr. Rudolf Schilder at Penn State University Department of Entomology to analyze cardenolide content in milkweed leaf samples. Dr. Daniel Swale at Louisiana State University, Department of Entomology, is helping to measure the effects of neuronal function in monarchs after direct exposure to cardenolides and pyrethroids. How have the results been disseminated to communities of interest?The results have been disseminated by presenting posters at multiple scientific societies meetings, including American Chemical Society (ACS), Society of Environmental Toxicology and Chemistry (CETAC), and Entomological Society of America (EntSoc). Furthermore, Co-PD Weissling and Ph.D. student Krueger, attended the Monarch Join Venture Partner's Meeting and engaged in the Monarchs around Agriculture working group, and engaged with the North American Pollinator Protection Campaign (NAPPC) Monarch Task Force and assist with preparing communication material. What do you plan to do during the next reporting period to accomplish the goals?Under each objective, we plan to perform the following activities to accomplish the goals. Objective 1. Surveys for monarch butterfly larvae and other endemic arthropods found on milkweed plants will be conducted across Nebraska in 2020. Objective 2. Estimate chronic LC10 and LC50 for the insecticide bifenthrin and the fungicide propiconazole. The Lowest Observed Effect Concentration (LOEC) will also be estimated from these experiments. The LOEC will be used to evaluate the effects of bifenthrin, propiconazole, and their combination, on monarch population growth parameters. Objective 3. Explore additional combinations of ouabain and bifenthrin, as well as underlying mechanisms of observed changes in detoxification enzyme activity. Determine the relationship between the pyrethroid-mediated reduction of hemolymph pH and the esterase-related detoxification of pyrethroid insecticides in monarch caterpillars as a pre-requisite to better understanding the physiology of this beneficial insect towards insecticide exposures. Objective 4. Design, collect, and process samples for RNA-sequencing of monarch larvae exposed to commonly used agricultural pesticides in Nebraska to characterize changes in gene expression and potentially identify stressor specific biomarkers.

Impacts
What was accomplished under these goals? Under goal 1, objectives 2, 3, and 4 of this project are encompassed. Objective 2. Rearing and artificial diet bioassays were standardized. Monarch population growth parameters were compared between an artificial diet with incorporated tropical milkweed powder and a tropical milkweed leaf diet to quantify population growth parameter differences when using an artificial diet for bioassays. The larval survival on the two diets was statistically similar. The total average development time of monarchs reared on the artificial diet was 5.5 days longer than leaf fed monarchs. Monarchs reared on the artificial diet had higher pupa weights than those raised on leaf fed diets. Cages for adult monarch mating pairs used for observations of adult parameters have resulted in premature adult mortality and diminished egg-laying. Current efforts are focused on improving adult cages to allow for accurate observation of adult survival and egg-laying. Objective 3. The effects of bifenthrin and ouabain exposure on caterpillar survival, weight gain, diet consumption, and enzyme activities were examined. The results of this experiment indicated that the combination of ouabain and contact exposure to bifenthrin did not change the LD50 of bifenthrin, but exposure to either ouabain or bifenthrin significantly affected caterpillar growth parameters (i.e., weight and diet consumption). The effects of bifenthrin and ouabain mixtures on caterpillar survival, total ATPase, general esterase, and cytochrome P450 monooxygenase were also examined. Total ATPase activity was reduced by exposure to bifenthrin, ouabain, and ouabain + bifenthrin. Whereas, high concentrations of ouabain significantly increased esterase activity after exposure to low levels of bifenthrin. Cytochrome P450 activity was significantly elevated with coexposure of ouabain and bifenthrin at the Lowest Observed Effect Concentration (LOEC). Another study compared the effects of pyrethroid insecticides, bifenthrin, and β-cyfluthrin, on the general esterase activities and hemolymph pH of fifth monarch caterpillars following acute topical treatments. Results of this study indicated that monarch caterpillars treated with 10 µg/ml of β-cyfluthrin for ­<60 min have significantly different β-esterase activity compared to individuals without pyrethroid treatment. Additionally, the data show that monarch caterpillars treated with 1, 3, and 10 µg/µl of bifenthrin for ­15 - 60 min. have significantly reduced hemolymph pH compared to individuals without pyrethroid treatment. Under goal 2, objective 1 of this project is encompassed. Objective 1. Surveys for monarch butterfly larvae and other endemic arthropods found on milkweed plants were conducted across Nebraska. A total of 7,234, 5,563, and 4,662 milkweeds (Asclepiasspp.) plants were surveyed in 2017, 2018, and 2019, respectively. Patches of milkweed (14 different species) included in surveys were selected from variable habitats along roads, adjacent to agricultural areas, and in state parks or federal lands. Sites sampled totaled 707 in 2017, 447 in 2018, and 290 in 2019. All plants or a subsample of plants in a patch were carefully inspected, and arthropods on plants were counted, and their numbers recorded. GPS coordinates of each sampling location were also recorded. In addition, the diversity of flowering plants and overall plant diversity was estimated at several sampling locations. Plants or crops in land adjacent to sampling locations were also noted. A total of 15, 79, and 90 monarch butterfly larvae were observed in 2017, 2018, and 2019, respectively. Monarch caterpillars were observed feeding on common, showy, sand, green comet, swamp, and spider milkweed species. Larvae were most commonly encountered in common and sand milkweed. Additional arthropods observed included several species of predators, herbivores, and a variety of pollinators. A correlation analysis between monarch caterpillar abundance, and sampling location as well as landscape ecology, land use patterns, and the abundance of flowering plants will be conducted when more data is collected. The same analysis will be conducted with beneficial insect and pollinator abundance and diversity.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: (Poster) Society of Environmental Toxicology and Chemistry 2019 General Esterase Activity of Monarch Butterflies (Danaus plexippus) Exposed to Pyrethroid Insecticides Haley Moyer*, Annie Krueger*, Tom Weissling, Ana Velez, and Troy D. Anderson
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: (Poster) Entomological Society of America 2019 Toxicology of a pyrethroid insecticide in the monarch butterfly (Danaus plexippus) and interactions with host plant defense chemicals and chemical fertilizers Annie J. Krueger*, Haley L. Moyer*, Tom Weissling, Ana Mar�a V�lez, and Troy D. Anderson
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: (3-minute Talk) Entomological Society of America 2019 Monarchs on the farm: How milkweed, insecticides, and potassium affect detoxification in the monarch butterfly Annie J. Krueger*, Terence A. Spencer, Tom Weissling, Ana Mar�a V�lez, and Troy D. Anderson
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: (Poster) Entomological Society of America 2019 Evaluation of Artificial Diet on Monarchs (Danaus plexippus L.) Population Growth PArameters for Pesticide Bioassays". Matthew Greiner*, Annie Krueger*, Terence A. Spencer, Troy Anderson, Thomas Weissling, Ana M. Velez
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: " (Poster) American Chemical Society 2019 Toxicology of a pyrethroid insecticide in the monarch butterfly and interactions with host plant defense chemicals Annie J. Krueger*, Haley L. Moyer*, Tom Weissling, Ana Mar�a V�lez, and Troy D. Anderson
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: (Poster) Society of Environmental Toxicology and Chemistry 2019 Toxicology of two pyrethroid insecticides in the monarch butterfly (Danaus plexippus) and interactions with host plant defense chemicals and potassium fertilizers Annie J. Krueger*, Haley L. Moyer*, Tom Weissling, Ana Mar�a V�lez, and Troy D. Anderson

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

Outputs
Target Audience:The target audience reached during this reporting period included the general public and undergraduate students from Insect Science, Biology, Environmental Restoration Science and Environmental Studies majors. Changes/Problems:Experiments to identify sublethal concentrations, carboxylase activity and gene expression, and collection of samples for RNA-sequencing were delayed due to issues establishing the monarch colony. The colony is now producing enough eggs to begin the testing in Spring 2019. What opportunities for training and professional development has the project provided?The project has provided training and professional development to two undergraduates and graduate students. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we plan to accomplish the following: Objective 1. Characterize habitat patches and landscape spatial arrangements in Nebraska and correlate with monarch larval abundance, surveys for monarch butterfly larvae and other endemic arthropods found on milkweed plants. Surveys for monarch larvae and other endemic arthropods on milkweed plants will continue to be performed in summer 2019. Objective 2. Measure the fitness costs of common stressors (i.e., insecticides, herbicides, and starvation) experienced by monarch larvae in agricultural environments. Insecticides concentration ranges and the effect on survival of field-relevant concentrations of a fungicide and herbicide on monarch larvae will be evaluated. Furthermore, the pesticide residue analysis performed on milkweed plants collected in the summer of 2018 will be evaluated. Objective 3. Measure the effect of pesticide exposure on monarch larvae lipid metabolism and function Standardization of the methods to evaluate the effect of pesticide exposure on monarch larvae lipid metabolism and function will be started. Objective 4. Perform RNA-sequencing of monarch larvae exposed to common agricultural stressors respectively. Samples of monarch larvae exposed to insecticides, a herbicide, and a fungicide will be collected, processed and sent for RNA-sequencing.

Impacts
What was accomplished under these goals? Goal 1 encompasses objectives 2, 3 and 4. Under objective 2 aimed to measure fitness costs of common stressors (i.e., insecticides, herbicides, and starvation) experienced by monarch larvae in agricultural environments, preliminary concentration ranges were identified using the surrogate species Spodoptera frugiperda. A surrogate species was used given that the colony was not producing enough eggs to perform the testing. The evaluation of the identified concentration ranges is planned to be evaluated in the Spring of 2019 besides the initiation of objectives 3 and 4 aimed to measure the effect of pesticide exposure on monarch larvae lipid metabolism and function and perform RNA-sequencing of monarch larvae exposed to common agricultural stressors respectively. Goal 2 encompasses objective 1 of this project. Under this objective of characterizing habitat patches and landscape spatial arrangements in Nebraska and correlate with monarch larval abundance, surveys for monarch butterfly larvae and other endemic arthropods found on milkweed plants were conducted across Nebraska. A total of 7,234 and 5,563 milkweeds (Asclepias spp.) plants were surveyed in 2017 and 2018, respectively. Patches of milkweed (12 different species) included in surveys were selected from variable habitats along roads, adjacent to agricultural areas, and in state parks or federal lands. Sites sampled totaled 707 in 2017, and 5564 in 2018. All plants or a subsample of plants in a patch were carefully inspected and arthropods on plants were counted and their numbers recorded. GPS coordinates of each sampling location were also recorded. In addition, the diversity of flowering plants and overall plant diversity was estimated at several sampling locations. A total of 15 and 79 monarch butterfly larvae were observed in 2017 and 2018, respectively. Monarch caterpillars were observed feeding on common, showy, sand, swamp, and spider milkweed species. Larvae were most commonly encountered on common and sand milkweed. Additional arthropods observed included several species of predators, herbivores, and pollinators. A correlation analysis between monarch caterpillar abundance, and sampling location as well as landscape ecology, land use patterns, and the abundance of flowering plants will be conducted when more data is collected. The same analysis will be conducted with beneficial insect abundance and diversity.

Publications