Source: UNIVERSITY OF MISSOURI submitted to NRP
DEVELOPING INNOVATIVE IPM TACTICS FOR BILLBUG CONTROL IN URBAN ENVIRONMENTS THROUGH EARLY DETECTION AND NON-CHEMICAL APPROACHES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1014224
Grant No.
2017-70006-27274
Cumulative Award Amt.
$321,957.00
Proposal No.
2017-04700
Multistate No.
(N/A)
Project Start Date
Sep 1, 2017
Project End Date
Aug 31, 2021
Grant Year
2017
Program Code
[ARDP]- Applied Research and Development Program
Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
(N/A)
Non Technical Summary
In the United States, managed turf, including residential, commercial and institutional lawns, parks, golf courses, and athletic fields, occupies more than 40 million acres of land, which are three times more acreage than does irrigated corn. To sustain green, dense, and pest-free turf surfaces, homeowners spend $60-65 billion dollars each year, representing a substantial increase since 1974 when $4 billion dollars were spent. Tremendous amount of resources, including 35,000 tons of pesticides, are applied to turf surfaces each year by homeowners and commercial turf managers, and some of which unfortunately, become source of contaminations of stream water and shallow ground water in urban areas.Recently, billbugs (Sphenophorus spp.) are increasingly being recognized as a serious threat to managed turf across North America. Reports from several regions reveal a billbug species complex that contains novel species and unique, complicated population dynamics, presumably resulting from climate changes and increased use of cold-tolerant warm-season turf species, such as bermudagrass (Cynodon spp.) and zoysiagrass (Zoysia spp.) in regions formerly dominated by cool-season turf species. Consequently, intensive billbug damage can occur at an alarming rate/scale. In response, turfgrass managers often use insecticides prophylactically, reflecting a lack of knowledge of the seasonal biology of this insect complex, and risk-averse behavior that is common among turfgrass managers. These chemical tactics mainly take the form of pyrethroid insecticides applied to kill billbug adults through contact or ingestion, or plant systemic insecticides (neonicotinoids or anthranilic diamides) applied to kill larval billbugs inside plant tillers, stolons and rhizomes. Both pyrethroids and neonicotinoids, that are favored classes of chemistry for billbug management, are orders of magnitude more toxic to important pollinators, such as honey bees, and natural enemies, such as ground beetle, than other classes of insecticides. This proposed project, therefore, aims to reduce our reliance on pesticide usage in the urban environment through better understanding of billbug seasonal biology and development of non-chemical control methods. Specifically, our objectives were to 1) validate a degree-day model to provide us a decision-making tool for taking actions, 2) determine the seasonal biology of billbug larvae using genetic tools, and 3) develop a mechanical tool using a professional turf sweeper to sweep and remove billbug adults, and ultimately control the pest population without heavy dependence on conventional insecticides. To achieve these objectives, a number of field and laboratory experiments will be conducted in Utah, Missouri, and Indiana. In Utah and Missouri, PDs Ramirez, Barrett, and Xiong will conduct a two-year field experiment to validate a previously developed degree-day model. This will involve treatments with two insecticide application timings based on degree-day model targeting 30% or 50% of adult activities, a traditional insecticide application timing based on calendar, and an untreated control. Billbug populations will be monitored using pitfall traps and soil samples for both adults and larvae populations, respectively, in order to validate and possibly fine tune the model. In Indiana, Co-PDs Richmond and Enders will validate and use the genetic tool previously developed to identify billbug larvae population collected from the three states to species. Larvae population will also be collected from April to October from all three states, and their species and seasonal phenology will be determined using this genetic tool and by measuring head capsule widths, respectively. In Missouri, PDs Xiong and Barrett will also perform a two-year field experiment to develop a mechanic tactic for billbug control by using a professional turf sweeper in the evening hours when billbug adult are active on the turf surface. This approach will be compared with insecticide applications and an untreated control, for their effectiveness in billbug control by using pitfall traps. Soil samples will be collected through the growing season for monitoring larvae population. Turf performance, influenced by treatment applications, will also be determined using visual assessment as well as sensor-based technology. Treatment effect on environmental safety will also be determined using a beneficial species, ground beetle, as an indicator. This will be carried out by collecting soil cores from treated area, and exposing ground beetles to the pre-treated soil cores and evaluating their survival and/or level of intoxication at hours and days after exposure. With completion of these objectives, we will deliver the knowledge gained through this project to the end-users by various field days, presentations at regional and national conferences, and communication/education through social media and website publications. With success of this project, we expect that professional turf managers will adopt this novel mechanical approach to control this pest, and ultimately reduced our dependence on pesticides.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110104010%
2150599106030%
2112130106030%
2162130106030%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants; 215 - Biological Control of Pests Affecting Plants; 216 - Integrated Pest Management Systems;

Subject Of Investigation
2130 - Turf; 0599 - Recreational resources, general/other; 3110 - Insects;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems);
Goals / Objectives
The proposed research seeks to develop innovative, sustainable approaches for the management of billbug in the urban environment using a mechanical tactic. Billbug damage to turf in many parts of the United States has become more problematic in recent years due to the emergence of a nationwide collage of billbug species. Novel species interactions have resulted in the development of regional billbug pest complexes, with poorly understood seasonal biology. The lack of a decision-making tool, both for adult and larval populations, has resulted in heavy reliance on prophylactic and often misguided insecticide applications. This unprejudiced insecticide application not only resulted in poor control of billbug population, but also increased risk of environmental safety to human, natural enemies, and beneficial species like pollinators, as well as elevated economic burden for professional turfgrass managers. Therefore, this project seeks to minimize insecticide uses to less amount/frequency, with specific objectives to:To validate a degree-day model that predicts the emergence and activity of adult billbugs;To determine the seasonal biology of billbug larvae through the use of a DNA-based tool;To develop and test a mechanical control approach using a professional turf sweeper, based on degree-day models, and determine the economic and environmental benefits of this strategy in comparison with conventional insecticide application.
Project Methods
Objective 1, validating a degree-day model for billbug species complex in this region, will be carried out at both Missouri and Utah for two growing seasons in 2018 and 2019. At each site, field experiment will be conducted with four treatments, including an untreated control, traditional management approach without degree-day model with insecticide bifenthrin, and management with the same insecticide based on updated degree-day parameters at 30% or 50% billbug activity benchmarks. Treatments will be arranged in a randomized complete block design with four replicates, with individual plot measuring 6 × 6 m with a 6 m boarder. The Intermountain West model for billbugs has been added to app for automated degree-day calculations, and the app provides an easy access to select the nearest/on-site weather-station to calculate degree-days. Treatment implementation and evaluation will follow established protocol by Co-PD Ramirez (Dupuy et al., in review). Briefly, pitfall traps will be installed in each plot to monitor adult billbugs pre- and post- management for determining adult suppression. Soil cores will be taken pre- and post- management to determine larval numbers and suppression. Sampling duration will be dependent on species and population activity associated with each state. For example, the Intermountain West billbug species complex appears to follow a single peak throughout the season, therefore, sampling will take place during this activity range. In Missouri, since two adult activity peaks have been observed, the sampling time will cover both spring and fall peak activity times. Objective 2, determining the seasonal biology of billbug larvae through the use of a DNA-based tool, will be mainly carried out by Co-PDs Richmond and Enders between November, 2018 and October, 2020, with billbug specimens collected from all three states. Briefly, during the first year of the study, adult specimens of each billbug species collected in Missouri and Utah (n=10 of each species from each location) will be shipped to the Co-PDs in Indiana (Richmond and Enders). Additional adults of each species will also be collected from Indiana. Genomic DNA will be extracted from whole-body homogenizations of adult specimens and quantified. Three loci commonly used for taxonomic identification, the mitochondrial cytochrome oxidase c subunit 1 gene (COI), ribosomal 18S subunit gene (18S), and the ribosomal second internal transcribed spacer region (ITS2), will be amplified using the primers and PCR conditions previously determined. Purified PCR products from each locus will be sequenced, and the resulting short read sequences will be mapped back to known reference sequences generated from our initial data set of Sphenophorus adults from Indiana. We will then use genetic distance and phylogenetic based methods to assess levels of sequence variation within and between Sphenophorus species at each locus (COI, 18S, ITS2) and evaluate the reliability of our DNA-based approach to identify billbug species across a broad geographic range. All outputs will be edited and annotated to construct a single tree grouping all billbug species collected from all locations. Additionally, larval populations will also be sampled and quantified at all states by weekly extracting 10 soil. Weekly collections will be placed into glass vials containing 95% ethanol, and shipped to Co-PD Richmond and Enders where head capsule widths will be measured, prior to determine the identity of each larval specimen using the genetic tool described above. Results will then be used to reconstruct the seasonal phenology of the larval populations established at each site. Objective 3, developing a mechanical control approach using professional turf sweeper, will be primarily performed in Missouri during the growing seasons of 2019 and 2020. Briefly, treatments will include mechanical removal of billbug adults using Professional Turf Sweeper ("mechanical method"), application of insecticide in the evening hours when billbug adults are active on the turf surface ("novel use of chemical"), application of insecticide during the normal daytime ("conventional chemical method"), in addition to a non-treated control ("Control"). All treatments will be arranged in a randomized complete block design with four replications. Individual plots, measuring 9m × 30m, will be established 20 meters apart from each other. Each plot will be installed with twenty pitfall traps and weekly monitoring will start at the beginning of February in 2019. The trapped billbug specimens, along with other insect species, will be transferred to the laboratory where they will be sorted and identified to species. Data collected from the pitfall traps will be utilized to fit the degree-day model and Co-PD Ramirez will assist in prediction the onset of 30% adult active time, indicating treatment application time. The mechanical method, using the LitterKat® Professional Turf Sweeper, will be performed in the evening hours after 9pm, during periods of peak adult active times in the spring, and again in the fall. Sweeping will be performed six times in spring (3 times/week for 2 weeks) and nine times in fall (3 times/week for 3 weeks). The collected billbugs and other insect species, likely spiders and ground beetles (Harpalus pennsylvanicus Degeer.) based on our experience, will be bagged per plot and transferred to the laboratory for identification. Treatment for novel use of chemical will apply insecticide bifenthrin at label rate in the evening hours after 9pm once in spring and once in fall when the degree-day model predicts the onset of 30% adult active time. Treatment with conventional method will use the same insecticide applying at the same timing, except that the application will be performed in the daytime as normal practice. Billbug larvae will also be sampled through 10 random soil cores from each plot once in summer following the oviposition of spring adult populations after overwintering, and once in fall before the large-sized larvae burrow down to deeper soil profile for overwinter. Turf performance, including percent injury (%), turf quality and normalized difference vegetation index (NDVI) will be performed once every two weeks during the two growing seasons.During the two-year experiment, treatment impact on beneficial species will also be assessed by exposing ground beetle, a natural enemy of billbug, to soil cores from each plot. Ground beetles will be collected using black light traps and/or evening captures using flashlights, and preserved in dark under room temperature with source of water and food. Within 24 hours after insecticide application, 10 ground beetles will be released to each of 6 enclosed soil cores randomly collected from each plot. Exposure of ground beetle to pre-sprayed soil cores, in comparison with non-treated controls, will be evaluated at 4 h, 1d, 3d, and 7d based on their movement and responses to light, and group them in category of dead or moribund, intoxicated, or normal following procedures described in Kunkel et al. (2001). Plots that received sweeping are expected to have no toxicity to ground beetles, although they might be picked up by sweepers which will be recorded for further analysis as well. Treatment residue effect on billbugs will also be evaluated based on a previously established procedure in PD-Xiong's lab. Briefly, 4 soil cores will be collected from treated plots randomly at 1, 2, 3, and 4 weeks after treatment application. Ten billbug adults will be released to each enclosed soil cores once collected, and the survival of billbugs will be evaluated in 48 h after carefully broken soil apart.All data collected will be subjected to ANOVA using the Proc Mixed procedure in SAS 9.4 (SAS Institute, Cary, NC). Mean separations will be carried out by Fisher's Protected LSD at P = 0.05.

Progress 09/01/17 to 08/31/21

Outputs
Target Audience:The target audiences are our students, both graduate and undergraduate students, other researchers and scientists, stakeholders and practitioners that include golf course superintendents, lawn care professionals, sod producers, groundskeepers, trade association members, and green industry representatives. The target audience also includes concerned public as urban environment affects majority of the population in the United States. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?At the University of Missouri, one graduate student (Matthew Fleetwood) and two undergraduate student researchers were involved. At Utah State University, Desiree Wickwar, an MS student was trained in conducting this project. At Purdue University, a graduate student (Marian Rodriguez Soto) has received advanced training in DNA extraction and sequencing techniques, bioinformatics and statistical analysis. She graduated in June 2021. How have the results been disseminated to communities of interest?A peer-viewed publication based on results from Objective 2 was generated and published in 2021. Three presentations based on this project were delivered in 2021 as well. The first was an invited webinar at the Golf Industry Show, which was an annual event organized by the Golf Course Superintendents Association of America. The second was a joint virtual field day between the University of Missouri and the University of Arkansas in August. In October, this research was also presented in the CPPM/ARDP Project Directors Workshop. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objectives 1 and 2 were previously completed and described. Objective 3 had three components: The first was to investigate the control of billbugs using a mechanical sweeper on zoysiagrass fairways traditionally infested with billbugs. At the University of Missouri, field experiments continued on plots that were established on a zoysiagrass (Zoysia japonica Steud.) at the Columbia Country Club in Columbia, Missouri, where a natural infestation of billbugs has been identified. Individual plots measured 6×10 m2 with 10 m border between plots. In each plot, 8 pitfall traps were installed and maintained year-round below the mowing line. Treatments consist of mechanical sweeping in the evening hours between 9:30 and 10:30 pm during the adult peak times in spring and fall, and applications of the insecticide bifenthrin in daytime (around 5pm) or night time (around 10pm) once on May 7, 2021 and once on September 3, 2021 as well as an untreated control. All four treatments were arranged in a randomized complete block design with four replications. In 2021, sweeping occurred 6 times from the end of May through June and 9 times between the end of August and mid-September. Adult billbugs showed two activity peaks with one in spring and one in early fall, similar to previous results. The highest hunting billbug counts in a single sweeping event was found in early fall where 103 individuals were collected in the sweeper. From the pitfall traps,the 8 pitfall traps in the untreated plots averaged 249 hunting billbug adults over the course of the growing season. In comparison, plots that were subjected to sweeping resulted in 153 billbugs over the season, which was 39% fewer billbugs than the control. Statistically the same number of billbugs were collected from the insecticide-treated plots, 100 and 105 for daytime or nighttime application, respectively. Considering that the plot area occupied by the 8 pitfall traps was only 0,5% of the each plot area, the real population of the hunting billbugs could be much higher. These results directly impacted turf quality. A high number of billbugs in the untreated plot resulted in zoysiagrass damage and the lowest turf quality (mean of 7.6 out of 9). As sweeping reduced the incident number of billbugs, turf quality increased to 8.0. Although the number of billbugs collected were similar for the daytime or nighttime insecticide application, there were significant differences in turf quality. Applying insecticide at night resulted in a similar turf quality to sweeping. However, turf quality averaged 8.2 in plots where insecticide was applied during the daytime. We hypothesize that many beneficial insects such as ground beetles that predate on billbugs and other insects like white grubs, are nocturnal and could have suffered as a result of applying insecticide at that time. On the other hand, daytime insecticide applications resulted in residual control of billbugs but did not interfere with the nocturnal activity of beneficial insects. A second component of Objecive 3 was to determine the residual activity of a soil-applied insecticide on billbug viability. At the University of Missouri, experiments were performed in the greenhouse to evaluate the residual effect of insecticides on the viability of hunting billbugs. The treatment (with or without bifenthrin) was applied to zoysiagrass turf in the field and soil cores (with grasses) were removed at 1, 2, 3, or 4 weeks after insecticide application and billbugs transplanted into containers sealed by cheese cloth. For each pot, 10 hunting billbugs were released into the pots and the billbug survival rates were determined after destructive sampling at 48 hours after exposure. The experimental design was a factorial combination of treatment (with or without bifenthrin) and sampling timing (1, 2, 3, or 4 weeks after insecticide application) with 3 replications. The entire experiment was repeated. Billbug viability increased step-wise throughout the sampling period, with 18, 40, and 60% viability for sampling conducted at 1, 2, and 3, weeks after insecticide application, respectively. The insecticide lost all effects after 4 weeks application, resulting 98% billbug viability. Because billbugs are prevalent throughout the growing season, limited insecticide residual suggests that repeated applications of insecticide are necessary for season-long suppression of billbugs. The third component of Objective 3 was identifying the safety of insecticide-treated soil cores to beneficial species such as ground beetles. Results were previously obtained and reported. Collectively, results from projects related to Objective 3 suggest that using a turf sweeper can effectively remove billbug adults in fall and spring when they are active on the turf surface. Our results also strongly suggest that mechanical removal of billbugs can enhance the overall turf performance, and play a significant role as a novel strategy for billbug management. Currently, we are in the process of developing a manuscript for a peer-reviewed publication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Rodriguez-Soto, M.M., L.S. Enders, R. Ramirez, X. Xiong, and D.S. Richmond. 2021. Characterizing billbug (Sphenophorus spp.) seasonal biology using NDA barcodes and a simple morphometric analysis. Insects. 12 (10), 930. DOI: 10.3390/insects12100930.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Barrett, B.A., M.E. Patterson1, and X. Xiong. 2018. Behavioral responses of hunting billbug (Coleoptera: Curculionidae) towards odors from different turfgrass species and adult conspecifics. Environmental Entomology. 47 (6): 1541-1546.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Rodriguez-Soto, M., L. Enders, L. Mason, R. Ramirez, X. Xiong, and D. Richmond. 2019. Characterizing billbug (Sphenophorus spp.) seasonal biology across different regions using DNA barcodes and simple morphometric analysis. Entomological Society of America. St. Louis, MO, November, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Rodriguez-Soto, M., L. Enders, R. Ramirez, X. Xiong, K. Umeda, and D. Richmond. 2019. A single mitochondrial gene (CO1) and simple morphometric measure provide insight into the seasonal biology of the billbug complex infesting turfgrass in North America. National Turfgrass Entomology Workshop, West Lafayette, IN, October, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Rodriguez-Soto, M., L. Enders, L. Mason, R. Ramirez, X. Xiong, K. Umeda, and D. Richmond. 2019. Development of a molecular tool for billbug larval identification using DNA barcodes. Entomological Society of America North Central Branch Meeting. Cincinnati, OH, March, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Xiong, X. 2020. Mechanical control for billbugs. 2020 North Central Extension & Research Activity 221 (Turfgrass and the Environment) Annual Meeting. Virtual. August 6, 2020
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Rodriguez-Soto, M., L. Enders, R. Ramirez, X. Xiong, and D. Richmond. 2020. Tracking billbug (Sphenophorus spp.) seasonal biology with DNA barcodes and a simple morphometric analysis to inform management. 2020 Entomology Society of America Annual Meeting. Virtual. November 16-19, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Xiong, X. 2021. Developing a novel mechanical strategy for control of billbug. 2021 Golf Industry Show. Webinar. February 2-4, 2021.
  • Type: Other Status: Published Year Published: 2021 Citation: Xiong, X. 2021. Developing a novel mechanical strategy for control of billbug. CPPM/ARDP Project Director Workshop. Webinar. October 13, 2021.
  • Type: Other Status: Published Year Published: 2021 Citation: Barrett, B., and X. Xiong. 2021. Discover the world of hunting billbugs. Joint Virtual Field Day between the University of Missouri and University of Arkansas. August 12, 2021.


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:The target audiences are our students, both graduate and undergraduate students, other researchers and scientists, stakeholders and practitioners that include golf course superintendents, lawn care professionals, sod producers, groundskeepers, trade association members, and green industry representatives. The target audience also includes concerned public as urban environment affects majority of the population in the United States. Changes/Problems:We have requested a no-cost extension of this project and after approval, our new expiration date for this project is August 31, 2021. Due to the unexpected pandemic starting this spring and the disturbances following the shut-down of the university campus and restricted access to office, lab and farm facilities, we experienced a delay in various activities associated with this project. As such, a one-year extension has been requested and granted, and with this extension, we'll be able to accomplish the proposed goals regarding this project. What opportunities for training and professional development has the project provided?At the University of Missouri, a graduate student, Matthew Fleetwood, continues to receive training to perform activities related to billbug collections and identification. An undergraduate student researcher has also been involved in this project. Desiree Wickwar, a MS student at Utah State University, is advised by Ramirez (co-PI). Ms. Wickwar has been trained to evaluate billbugs and predators that may impact their management. Currently, Ms. Wickwar is heavily involved in learning experimental design, data collection, and analysis. In addition, Ms. Wickwar is adding to her professional development by giving presentations on her research. In 2019, Ms. Wickwar received a 1nd place award for her presentation at the national Entomological Society of America conference in St. Louis, MO. At Purdue University, a graduate student working primarily on objective 2 (Marian Rodriguez Soto) has received advanced training in DNA extraction and sequencing techniques, bioinformatics and statistical analysis. She continues to presented her results at various scientific and extension events with a planned graduation date of June 2021. How have the results been disseminated to communities of interest?At the University of Missouri, a presentation was given at the 2020 North Central Extension and Research Activity 221 Annual Meeting (virtual meeting) to members in the turf academic community in this region. We received very positive feedback from the audience, and consequently, have been invited to give a presentation to the upcoming 2020 Golf Industry Show (virtual meeting) in February of 2021. At Utah State University, a presentation has been made based on results from this project at the Utah Green Conference, which occurred on January 27, 2020. Results have also been dissipated to the audience at the national ESA annual meeting in November 18, 2019, at St. Louis, MO by co-PI Ramirez and his team members. Results coming from Objective 2 have been disseminated in the past year at several scientific (Entomological Society of America, North Central Branch of the Entomological Society of America) and extension (Turf & Landscape field, Indiana Green Expo, Professional Landscape Management School) venues. What do you plan to do during the next reporting period to accomplish the goals?At the University of Missouri, we plan to continue the field sweeping experiment in 2021, as well as complete our identification of the billbugs collected from both biology and sweeping experiments during the 2020 season. We were not able to complete this task in 2020 due to lockdown of the university campus for most of the growing season. We would also like to repeat the ground beetle safety experiment in 2021.We also plan to summarize research findings in a couple of peer-reviewed article in the next year.

Impacts
What was accomplished under these goals? Ojective 1 was to gain knowledge on pest biology and seasonal dynamics of the pest complex, and validate a degree-day model. Activities associated with this objective were performed at both Utah and Missouri, which included establishing field plots, collecting billbug specimens from field plots, and gathering environmental data for degree-day model validation. At Missouri, 40 pitfall traps were installed at the Columbia Country Club in Columbia, Missouri, and weekly data collection has started since spring of 2018 and continued into 2020. In 2018, we observed two adult activity peaks with one in spring between April and June, and one in fall between August and September. A similar pattern was found in 2019. In 2020, November 6 was the latest date that a hunting billbug was collected from the pitfall traps. As of now, we are still in the process of analyzing data collected from 2020, and are working with a statistician to validate the degree-day model that best fits our data. In Utah, the Utah-Idaho model predictions have begun to provide timings for preventive and curative applications of systemic, long-residual insecticides in billbug management. Objective 2 was to provide a reliable DNA-based approach for identifying billbug larvae that will allow us to accurately characterize the complete seasonal biology of the billbug complex across large geographic regions of the U.S. Billbug specimens were collected from a total of four states in 2018 and 2019, including Indiana, Missouri, and Utah and Arizona. Progress on this objective has provided a robust DNA-based approach for identifying billbug larvae to species level using CO1 alone. We are now able to differentiate all common billbug species and this tool appears to work well across geographic regions. Using larval specimens collected from Utah and Indiana we have also combined molecular data with a simple morphometric measure (head capsule diameter) to create seasonal phenology maps that clearly indicate differences in larval seasonal biology and species composition of the billbug complexes occurring at these locations. These maps are being incorporated into existing, revised extension materials and will be useful for designing and communicating regionally relevant IPM strategies. We continue to communicate these results at various scientific and extension venues. Objective 3, at the university of Missouri, field plots were established on a zoysiagrass (Zoysia japonica Steud.) at the Columbia Country Club in Columbia, Missouri where natural infestation of billbugs have been identified. Individual plots measured 6×10 m2 with 10 m boarder between plots. In each plot, 8 pitfall traps were installed and maintained year-round below the mowing line. Treatments consist of mechanical sweeping in the evening hours between 9:30 and 10:30 pm during the adult peak times in spring and fall, and applications of the insecticide bifenthrin in daytime (around 5pm) or night time (around 10pm) once on April 24th and once on September 9th, 2019, as well as an untreated control. All four treatments were arranged in a complete block design with four replications. In 2020, sweeping occurred 6 times between June to early July, and 9 times between August and September. Due to the shutdown of the university campus and consequently our inability to access the laboratory in 2020, we are still in the process of identifying collected billbugs to species. Similarly, we are still processing the billbugs collected from the 8 pitfall traps that are installed in each of the plots. Other data, including turf quality, NDVI, and percent turf injury due to billbug activities were collected as described for the previous year and currently the data are being analyzed. Additionally, we have also performed the insecticide residual effect on hunting billbugs in the greenhouse. Treatment (with or without bifenthrin) was applied to zoysiagrass turf in the field and soil cores (with grasses) were removed at 1, 2, 3, or 4 weeks after insecticide application and billbugs transplanted into containers sealed by heese cloth. For each pot, 10 hunting billbugs were released into the pots and the billbug survival rates were determined after destructive sampling at 48 hours after exposure. The experimental design was a factorial combination of treatment (with or without bifenthrin) and sampling timing (1, 2, 3, or 4 weeks after insecticide application) with 3 replications. The entire experiment was repeated once. We initiated this experiment in August of 2020, and currently, the data are still under analysis as of now. The environmental safety experiments with specific goals to evaluate how sweeping or insecticide applications affect beneficial species, such as ground beetle, were carried out in 2020 as well. Starting early August 2020, a series of growth chamber experiments were carried out in which plugs that received sweeping or insecticide application (bifenthrin) within 24 hours were collected and repotted in the growth chamber using sand. For each pot, 6 ground beetles were released into the pot, and during the 7-day period, water and food (dog food) were supplied in a small container on the surface of each pot. In the evening hours (9 pm), the status of the ground beetles were assessed with white light and gentle touching, when this nocturnal species was on the soil surface searching for food and/or water. This was carried out at 4 hours, or 1, 3, or 7 days after exposure. The ground beetles' reaction to light were categorized as normal, intoxicated, and dead or moribund based on Kunkel et al. (2001). A total of 6 runs/replications were carried out for this experiment. As expected, 100% of the ground beetles released to control pots appeared to be normal, regardless of time of exposure. At four hours after exposure, all ground beetles appeared to be normal under white light. In comparison, 2.4% of ground beetles exposed to the insecticide-treated plugs were intoxicated at 1 day after exposure. By 3 days after exposure, the percentage increased to 7.1% for intoxicated ground beetles, and an additional 1.2% were dead at this stage. By 7 days after exposure to the insecticide-treated plugs, the intoxicated ground beetles reduced to 2.4% but the dead ground beetles increased to 9.5%. This result indicated that nearly 1 out of 10 ground beetles would have been killed by application of bifenthrin after 7 days' exposure. Combining with the intoxicated ground beetles, the percentage increased to 11.9%, indicating nearly 12 out of 100 ground beetles would be expected to be killed or negatively impacted by the insecticide, despite additional supplies of water and food. As our data showed an exponential increase of combined intoxicated and dead ground beetles with prolonged exposure, we hypothesize that under field conditions without additional food and water supplies, a greater number of ground beetle would have been negatively impacted following insecticide applications as the bifenthrin can maintain at least 3-week soil residual effect based on our other studies. At Utah State University, a series of microcosms were setup to quantify the extent to which species richness of predators impacts billbug feeding behavior, and investigate resulting mechanisms. Billbugs were subjected to varied levels of predator presence (0 (control), 1 or 2 predator species) either in direct contact or indirect contact (separated by a screen). We found that billbugs responded to predator presence by decreasing feeding time compared to the no predator control. Even without direct contact, predators elicited decreased feeding. The magnitude of the response was increased with additional predator species present. The indirect nature of the response suggests predator odors may play a role.

Publications


    Progress 09/01/18 to 08/31/19

    Outputs
    Target Audience:The target audiences are our students, both graduate and undergraduate students, other researchers and scientists, stakeholders and practitioners that include golf course superintendents, lawn care professionals, sod producers, groundskeepers, trade association members, and green industry representatives. The target audience also includes concerned public as urban environment affects majority of the population in the United States. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?At the University of Missouri, a graduate student, Matthew Fleetwood, continues to receive training to perform activities related to billbug collections and identification. An undergraduate student researcher has also been involved in this project. Desiree Wickwar, a MS student at Utah State University, is advised by Ramirez (co-PI). Ms. Wickwar has been trained to evaluate billbugs and predators that may impact their management. Currently, Ms. Wickwar is heavily involved in learning experimental design, data collection, and analysis. In addition, Ms. Wickwar is adding to her professional development by giving presentations on her research. In 2018, Ms. Wickwar received a 2nd place award for her poster at the joint Entomological Society of America, and Entomological Society of Canada conference in Vancouver, BC. At Purdue University, a graduate student working primarily on objective 2 (Marian Rodriguez Soto) has received training in DNA extraction, sequencing and bioinformatics. She has presented her results at several venues. The student has also been directly involved in translating and disseminating her findings at our Turf and Landscape field day, with more than 500 in attendance. How have the results been disseminated to communities of interest?At the University of Missouri, a presentation was given at the University Turfgrass and Ornamental Field Day on July 30 to turf professionals. Some of the results from sweeping experiments (Objective 3) were discussed, and we have received very positive feedback from golf course superintendents and lawn-care professionals. At Utah State University, a presentation has been made based on results from this project at the Utah Green Conference, which occurred on January 28, 2019. Results have also been dissipated to the audience in ESA, ESB, and ESBC joint annual meeting n November 12, 2019, at Vancouver, BC by co-PI Ramirez and his team members. Results coming from Objective 2 have been disseminated at several scientific meetings (Entomological Society of America, Ohio Valley Entomological Society and National turfgrass Entomology Workshop) and our Turf & Landscape field day which regularly draws over 500 landscape management professionals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Billbugs are small-size weevils, which have increasingly being recognized as a serious threat to managed turf across North America. In response, turfgrass managers use insecticides prophylactically, reflecting a lack of knowledge of the seasonal biology of this insect complex, and risk-averse behavior that is common among turfgrass managers. This project aims to generate a better understanding of its biology, and to adopt an innovative use of a mechanical sweeper to remove this pest from the turf system. Expected outcomes from this project include a genetic tool to proper identify billbug species, a decision-making tool based on a deeper understanding of billbug biology, a novel control strategy using mechanical approach, and ultimately a sustainable, ecologically-based IPM system to control billbug in the urban environment. Success of this project will allow turfgrass managers to avoid excessive and inappropriate use of insectaries and in most cases, herbicides because insect damage on turf often leads to weed infestation. Ultimately, this project will lead to enhanced environmental safety for beneficial species, as well as human beings. For specific goals, objective 1 was to gain knowledge on pest biology and seasonal dynamics of the pest complex, and validate a degree-day model. Activities associated with this objective were performed at both Utah and Missouri, which included establishing field plots, collecting billbug specimens from field plots, and gathering environmental data for degree-day model validation. At Missouri, 40 pitfall traps were installed at the Columbia Country Club in Columbia, Missouri, and weekly data collection has started since spring of 2018 and continued into 2019. In 2018, we observed two adult activity peaks with one in spring between April and June, and one in fall between August and September. A similar pattern was found in 2019. The maximum billbugs collected during a one-week period was in the last week of May where 74 total billbugs were collected from the 40 pitfall traps. As of now we are working with a statistician to develop our degree-day model for predicting billbug occurrence and hence insecticide application timing based on Missouri's climate. This model will be validated in 2020. In Utah, the Utah-Idaho model predictions have begun to provide timings for preventive and curative applications of systemic, long-residual insecticides in billbug management. Objective 2 was to provide a reliable DNA-based approach for identifying billbug larvae and allow us to accurately characterize the complete seasonal biology of the billbug species. Billbug specimens were collected from all three states in 2018 and 2019, including Indiana, Missouri, and Utah. Progress on this objective has provided a robust DNA-based approach for identifying billbug larvae. Using CO1 alone, we are able to differentiate 5 common billbug species and this tool appears to work well across geographic regions. Using larval specimens collected from Utah we have combined molecular data with a simple morphometric measure (head capsule diameter) to create a seasonal biology map that will be useful for designing and communicating IPM strategies. This same approach will be used for larval specimens collected from MO. Results have been communicated in several venues during the past year. Objective 3 aims to develop an innovative IPM tactic using mechanical sweeper to reduce billbug population, and to assess the influence of such a practice to environmental safety by assessing beneficial species. At the university of Missouri, field plots were established on a zoysiagrass (Zoysia japonica Steud.) at the Columbia Country Club in Columbia, Missouri where natural infestation of billbugs have been identified. Individual plots measured 6×10 m2 with 10 m boarder between plots. In each plot, 8 pitfall traps were installed and maintained year-round below the mowing line. Treatments consist of mechanical sweeping in the evening hours between 9:30 and 10:30 pm during the adult peak times in spring and fall, and applications of the insecticide bifenthrin in daytime (around 5pm) or night time (around 10pm) once on April 24th and once on September 9th, 2019, as well as an untreated control. All four treatments were arranged in a complete block design with four replications. In 2019, sweeping occurred 6 times in spring between late April to early June, and 9 times between August and September. As of now, data collected in 2019 showed that throughout the 2019 season, the four replicated plots that received mechanical sweeping removed a total of 679 hunting billbugs with the maximum number of 103 hunting billbugs removed following a single sweeping event on August 19, 2019. The removal of hunting billbugs from these swept plots resulted in a significantly less billbugs collected weekly from the 8 pitfall traps, averaging 5.3 hunting billbugs throughout the entire growing season from mid-April to the beginning of November, compared to an average of 8.6 counts in the untreated control plots. This reduction of billbugs in the swept plots significantly improved the turf quality which was visually assessed weekly during the entire growing season, resulting an average of 8.0 rating out of a total 9 possible points, compared to control plots where the average turf quality was determined to be 7.6 throughout this year. All of the results described above were pooled over the entire growing season as no treatment by evaluation timing interactions were detected. In comparison, the insecticide treated plots resulted in the least amounts of billbugs collected form the 8 pitfall traps installed inside each plot, averaging 3.4 or 3.6 counts, for insecticides applied in daytime or night time respectively. These results suggested that by comparison, the insecticide (bifenthrin) we applied demonstrated a certain degree of soil residual activity; by comparison, billbugs outside of the control and swept plots were able to reenter during this experiment. Nevertheless, our data collected in 2019 showed strong evidence that mechanical sweeping is a promising approach for billbug control and had it been applied to the entire fairway, for example, it would have had dramatically reduced the billbug population without resorting to pesticides. The second-year experiment will be continued at the same location in 2020. At Utah State University, an evaluation of the impact beneficial predatory species has on billbugs was conducted. From an initial research article (Dupuy and Ramirez, 2019) it was determined that predatory ground beetles and spiders appear to have little direct consumption (5.97%) of adult billbugs and that larvae may escape predation as they move deeper in the soil profile. Based on this information, a subsequent study is being conducted to evaluate the indirect, non-consumptive effects of predators on billbugs given that billbugs engage in diverse behaviors when exposed to predators including thanatosis (playing dead) and burying their head in soil. An experiment was setup to evaluate the behavioral responses of adult billbugs to predatory ground beetles in direct contact, and predator odor (cotton swab exposed to predators for 72hrs) or indirect exposure to predators (separated by screen) in mesocosms. An additional positive control in which billbugs were indirectly exposed to a non-predatory arthropod (separated by screen) was used. Initial results suggest that indirect cues (volatile odors and visual) increased head burying behavior, yet decreased feeding behavior compared to the control. A series of Y-tube experiments are being conducted to evaluate predator odors on billbug behavior.

    Publications


      Progress 09/01/17 to 08/31/18

      Outputs
      Target Audience:The target audiences are our students, both graduate and undergraduate, other researchers and scientists, stakeholders and practitioners that include golf course superintendents, lawn care professionals, sod producers, groundskeepers, trade association members, and green industry representatives. The target audience also includes concerned members of the public, as the urban environment affects a majority of the population in the United States. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?At the University of Missouri, a graduate student, Michael Patterson, participated in this project in his last year of M.S. training and assisted with site establishment, billbug collection, and control. Mr. Patterson's M.S. thesis research focused on understanding the biology of hunting billbug, which is related to this proposed project. Starting in 2018, another M.S. student, Matthew Fleetwood, has been receiving training to perform activities related to billbug collection, identification, and control under field conditions. Though the Summer Research Opportunity Program (SROP) and Summer Bridge program, the CoPI's at Purdue have recruited and enrolled a graduate student, Marian Rodrigues Soto, to work on Objective 2. The goal of these programs is to recruit, mentor and prepare high quality students from under-represented minority groups in the U.S. During the summer, the student received training in modern DNA extraction, purification and sequencing techniques. She has also presented her preliminary findings in the form of a poster and oral presentation during the SROP/Bridge conference at Purdue (July 26, 2018) and as an oral presentation at the annual meeting of the Ohio Valley Entomological Association (OVEA) (Friday, October 19, 2018, Indianapolis IN). Madeleine Dupuy, a PhD student at Utah State University, contributed to the project in the last year of her graduate work. She was able to compile her data from pesticide trials and using the regional degree-day model, and submit a manuscript for publication. Desiree Wickwar, was recruited as an MS student at Utah State University, and continue with the completed work on billbugs from Dr. Dupuy. Ramirez (co-PI) is Ms. Wickwar's major advisor in the graduate program. Before completing her degree, Dr. Dupuy assisted in training Ms. Wickwar on billbugs and their management. Ms. Wickwar received training to identify billbug species and collect billbugs using linear pitfall traps and soil core sampling. How have the results been disseminated to communities of interest?The graduate student recruited by the CoPI's at Purdue presented her preliminary findings in the form of a poster and oral presentation during the SROP/Bridge conference at Purdue (July 26, 2018) and as an oral presentation at the annual meeting of the Ohio Valley Entomological Association (OVEA) (Friday, October 19, 2018, Indianapolis IN). The Purdue Graduate Bridge program offers a unique opportunity for incoming graduate students to take part in funded summer research and mentoring. Graduate Bridge is particularly interested in under-represented students who have been admitted to one of the STEM (science, technology, engineering or mathematics) disciplines. Students are nominated by their department for the program. Incoming graduate students accepted into the Graduate Bridge program conduct research over an eight-week period with their faculty mentor. This summer experience prepares students for the upcoming year at the University by providing professional development opportunities that assist them throughout their academic programs, giving them an instant peer network aimed at improving retention. The mission of OVEA is to encourage and cultivate the professional development of students through continued sponsorship of an Annual Forum. The forum aims to promote the study of entomology as a science, reward student excellence in academic achievement and improve public awareness and understanding of the science of entomology. At the Utah State University, information regarding billbug control timing is summarized in an extension publication. Also the CoPI at Utah presented related research findings in both SERA IEG 25/ WERA IEG 11 Joint Annual Meeting at Ft. Lauderdale, FL, and USU Turf Field Day at Logan UT. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

      Impacts
      What was accomplished under these goals? -- In the United States, managed turf, including residential, commercial and institutional lawns, parks, golf courses, and athletic fields, occupies more than 40 million acres of land. For perspective, turfgrass occupies three times more acreage than does irrigated corn. To sustain a dense, green and pest-free surface, Americans spend more than $60 billion dollars annually, and apply over 35,000 tons of pesticides to the turf. Unlike other agricultural land, turf is a prominent component of people's day-to-day life. This probably explains the deep concern that the general public has over the environmental risks of managed turf to human beings, as well as domestic and wild animals in the urban area. The objective of this project, therefore, was to minimize our dependence on pesticide use on turf for control of billbugs (Sphenophorus spp.). Billbugs are small-size weevils, which are increasingly being recognized as a serious threat to managed turf across North America. In response, turfgrass managers use insecticides prophylactically, reflecting a lack of knowledge of the seasonal biology of this insect complex, and risk-averse behavior that is common among turfgrass managers. This project aims to generate a better understanding of its biology, and to adopt an innovative use of a mechanical sweeper to remove this pest from the turf system. Expected outcomes from this project include a genetic tool to proper identify billbug species, a decision-making tool based on a deeper understanding of billbug biology, a novel control strategy using mechanical equipment, and ultimately a sustainable, ecologically-based IPM system to control billbug in the urban environment. Success of this project will allow turfgrass managers to avoid excessive and inappropriate use of insectaries and in most cases, herbicides because insect damage on turf often leads to weed infestation. Ultimately, this project will lead to enhanced environmental safety for beneficial species, as well as human beings. For specific goals, objective 1 was to gain knowledge on pest biology and seasonal dynamics of the pest complex, and validate a degree-day model. Activities associated with this objective were performed in both Utah and Missouri, which include establishing field plots, collecting billbug specimens from field plots, and gathering environmental data for degree-day model validation. In Utah, we used two systemic insecticides, Acelepryn® and Merit®, and two bioinsecticides, Grandevo® and Venerate®, which contain bacteria and their fermentation products, to determine whether preventive and curative insecticide application timings predicted by the Utah-Idaho degree-day model were effective against Inter-mountain West billbugs. In addition, data from a pesticide applicator were used to determine how model-predicted management timings compare to traditional, calendar-based management strategies. We found that Acelepryn® was most effective against billbugs out of all products tested, especially at the preventive timing, while bioinsecticides were not effective at either timing. It is possible that a single application of bioinsecticides was not sufficient to provide billbug suppression. Utah-Idaho model predictions appeared sufficient to time preventive and curative applications of systemic, long-residual insecticides. Instances where a pesticide applicator treated within model-predicted timings were also effective. These data begin to provide validation of a degree-day model and its use in billbug management timing. In Missouri, one field site was identified and the field plots were established at the Columbia Country Club in Columbia, Missouri. Beginning in April, 2018, weekly billbug collections were performed from 40 pitfall traps installed. The billbugs collected were transported into the laboratory where identification and count of species were carried out. Temperature data were recorded for this period of time as well. For 2018, adult activity showed two peak periods. One peak period was found in spring between April and June, with the maximum billbugs (60+) found during a one-week period in mid-May. The second peak was found in fall between August and September. This trend was different from the pattern observed in Utah where only one peak of adult activity was found per year. The trend observed in 2018, however, was very similar to the trend recorded in the past year we have in Missouri, suggesting possibly two overlapping generations of hunting billbug in Missouri. With data collected in 2019, a region-specific degree-day model can be developed for predicting billbug occurrence and hence application timing for Missouri's climate. Objective 2 was to provide a reliable DNA-based approach for identifying billbug larvae and allow us to accurately characterize the complete seasonal biology of the billbugs species. Billbug specimens have been collected from all three states in 2018, including Indiana, Missouri, and Utah. At the University of Missouri, a billbug nursery was also established in 2018 by inoculating a fenced zoysiagrass plot with billbug adults collected from different sites throughout the year. The goal is to provide a sustainable supply for billbug larvae specimens in the next two years, since extracting billbug larvae is destructive to turf and is not feasible for commercial golf courses or sod farms. At Purdue University, adult billbugs from Indiana, Missouri, Utah and Arizona were collected and identified morphologically to species. Genomic DNA was extracted from the specimens, and cytochrome oxidase subunit 1 (CO1) was amplified and sequenced. The maximum likelihood tree resulting from sequence data showed species differentiation that was not influenced by geography, producing monophyletic clades of each species [S. ineaqualis (Indiana only), S. cicatristriatus (Utah only), S. phoeniciensis (Arizona only), S. parvulus (Indiana, Missouri and Utah), and S. venatus (Indiana, Missouri and Utah)] with bootstrap values of greater than 82%. These results imply that CO1 may be useful for resolving larval phenology across regions even when multiple species are present. We will continue to develop and fine tune our molecular identification tool in the coming year by focusing on two additional genetic loci (18S and ITS2). This tool promises to provide a reliable DNA-based approach for identifying billbug larvae. It will allow us to accurately characterize the complete seasonal biology of the billbugs species prevalent at each location and provide us with the ability to differentiate various billbug species, even when they occur in mixed species populations. Ultimately, completion of this objective will provide a foundation for the development of management programs that are based on billbug biology, rather than on calendar date. Objective 3 aimed to develop an innovative IPM tactic using mechanical sweeper to reduce billbug population, and to assess the influence of such a practice to environmental safety by assessing beneficial species. At Missouri, an experimental site with natural infestation of billbugs has been identified. The experiment will begin in 2019, and the field experiment will carried out in both 2019 and 2020.

      Publications

      • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Dupuy, M., A. VanDyke, and R.Ramirez. 2018. Evaluating recommended management timings against billbugs (Coleoptera: Dryophthoridae) using a regional Intermountain West degree-day model. Crop Forage and Turf Management (under review)
      • Type: Other Status: Published Year Published: 2018 Citation: Ramirez, R. 2018. Turf billbug management - Timing it right. Utah Pest Newsletter, Summer edition https://utahpests.usu.edu/files/up-newsletter/2018/UtahPests-Newsletter-summer18.pdf
      • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Rodrigues Soto, M., L. Enders, L. Mason and D. Richmond. 2018. Adult billbug (Coleoptera: Dryophthoridae: Sphenophorus Spp.) identification using DNA barcodes. Purdue SROP/Bridge Conference, July 26, 2018, West Lafayette, IN.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Rodrigues Soto, M., L. Enders, L. Mason and D. Richmond. 2018. Development of a reference database of DNA sequences from adult billbug species for larval identification. Annual Meeting of the Ohio Valley Entomological Association, October 19, 2018, Indianapolis IN.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ramirez, R. 2018. Turf insect management in the West. SERA IEG 25/ WERA IEG 11 Joint Annual Meeting, July 11-13. Ft. Lauderdale, FL
      • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Rodrigues Soto, M., L. Enders, L. Mason and D. Richmond. 2018 Adult billbug (Coleoptera: Dryophthoridae: Sphenophorus spp.) identification using DNA barcodes. Purdue SROP/Bridge Conference, July 26, 2018, West Lafayette, IN.