MEET THE DUNG BEETLES: IDENTIFYING COMMUNITIES AND ASSESSING THREATS TO DUNG BEETLES OF WV

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028089
• Grant No.: 2022-69014-36669
• Cumulative Award Amt.: $300,000.00
• Proposal No.: 2021-10985
• Project Start Date: Feb 1, 2022
• Project End Date: Jan 31, 2025
• Grant Year: 2022
• Program Code: [A1261]- Inter-Disciplinary Engagement in Animal Systems

Recipient Organization
WEST VIRGINIA UNIVERSITY
886 CHESTNUT RIDGE RD RM 202
MORGANTOWN,WV 26505-2742

Performing Department
Davis College of Agriculture

Non Technical Summary
Dung beetles perform valuable ecosystem services to pastures by decomposing manure, thus increasing nutrient cycling and combatting animal parasites. However, many dung beetles are harmed by chemical parasiticides used to treat livestock. To maximize pasture productivity and ecosystem services, we propose to investigate the dung beetle communities of WV and effective parasite control methods that protect dung beetles. Research on how parasite control products affect dung beetles has largely focused on a single class of dewormers: ivermectins. However, a survey of local cattle producers indicated that 50% of respondents were using other products, but we do not understand how toxic these other products are to dung beetles. Additionally, in WV, the current dung beetle species list includes only 13 species, while neighboring states can have 5x more species. Characterizing the dung beetle community provides a baseline for future studies on climate change, parasite management, and grazing practices. To support livestock in Appalachia, we propose the following three objectives: first, we will investigate the risk to dung beetles of the four most common parasite control products. Second, we will investigate the relationships between landscape, parasite treatments, and pasture management on dung beetles by surveying the dung beetles on livestock farms. Third, we will provide training for extension educators and establish a dung beetle ID service to which WV residents can send their dung beetles to be identified.Ultimately, we hope to promote the productivity and sustainability of pasture ecosystems and livestock production by understanding dung beetle communities in WV.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
121	1610	1130	50%
216	3310	1150	50%

Knowledge Area
216 - Integrated Pest Management Systems; 121 - Management of Range Resources;

Subject Of Investigation
3310 - Beef cattle, live animal; 1610 - Pasture;

Field Of Science
1130 - Entomology and acarology; 1150 - Toxicology;

Keywords

beetle id services

dung beetle

extension training

parasite management

pasture management

Goals / Objectives
Theaimof this initial seedgrant is to assess the relative risks of anti-parasitic drugs to common WV dung beetles, identify an initial baseline of cattle dung-feeding beetles across the state,provide information toWVpasture management expertsabout the ecology of these important species and the risks that they face,and engage livestock producers in their own dung beetle communities.Goals: (1)Assess riskofcommon internal and external parasitecontrolmethodstoWV dung beetles.Hypotheses:(1)Continuous release of Eprinomectin (Long-range®)will be the most toxic to dung beetles, followed bylong-term use ofDiflubenzuron(Clarifly).Fenbendazole(Safeguard)will be relatively non-toxic.(2)Toxicity will depend on beetle size -smaller beetles will be more sensitive(2)Investigate the relationships across WV between dung beetle abundance and richness, landscape, and herd management.Hypotheses:(1)Different parts of the state have a different community of beetles, thus the potential community in pastures will depend on region. Similarly, forested landscapes have a more abundant dung beetle community. Thus the potential dung beetle community for each farm will depend on geography and landscape cover.(2)Pasture management strongly affects dung beetles. Herds that have been managed for a long time with Ivermectinwill likely have a less speciose/abundant dung beetle community.Herdsthat are managedwithFenbendazolewill have amore diversedung beetle community.(3)Educate extension agents and engagestakeholders in dung beetle conservation with a Dung beetle ID service and extension training.Hypothesis: Knowing more about the dung beetle community on their land will alter perceptions about theimportance of dung beetles.This will encourage producers to use an IPM approach to parasite management that includesidentifying animals withparasitesandtreating animals that are most susceptible.

Project Methods
Objective1:Ourpreliminary survey indicated that Safeguard (dewormer-fenbendazole),Long-range(dewormer- Eprinomectin),Cydectin(dewormer/fly control- moxidectin), andClarifly(fly control-diflubenzuron) are commonly used in the region, and have not been as extensivelyinvestigatedas Ivermectin.Tocharacterize exposure of dung beetles to these products, we will measure fecal concentrations of active ingredients over time.We will apply Safeguard,Long-Range,Cydectin, orClariflytofiveyoungcowseach yearon the WVU J.D. Ruby Research Farm in Reedsville WV.Fresh dung will be collected in a time series 1 d, 3 d, 7 d, 14 d, 30 d, 60 d, and 90 d after initial treatment.Concentrationsof the active ingredients in dung samples will be quantified by HPLC following published protocols29,30, adapted to available instrumentation. Preliminary data confirms our ability to resolve the chemicals of interest, and readily detect them at low ppm concentrations in analyte preparations by photodiode array detection.These data will provide information about the timing of maximum concentration of product that beetles may be exposed toandis an essential component of assessing risk to dung beetles.To quantifytoxicityof these products on localdung beetle communities,wewill perform toxicity assays on four species of dung beetles,Onthophagus taurus(medium-sized,introduced),On.hecate(med.,native),On.pennsylvanicus(small,native),andOscarinusrusicola(small,native). Beetles will be field collectedfrom the WVU Animal Sciences farm,the WVU Organic Farm, and the WVU J.D. Ruby Research Farmin the springto establishcolonies for toxicity assay experiments.InYear 1 we will establish the initial range of toxicities for pairs of adult beetles. InYear 2, we will establish more preciselethal concentrations (LC50s).To test toxic doses on each pair of beetles,theactive ingredient will be incorporatedusing a mechanical stirrerintopreviously-frozen cow dung, which we willcollect during winter to ensure it hasnot been treated with any productsfor3 months31,32. Previous work indicates thattheLD50 forIvomectincan range from 0.016µg/g to 0.59 µg/g depending on species and life-stage.We will estimate LD50s inYear 1 usingfive log-scale does ranging from0.001 µg/gto10 µg/g manure, and an untreated control. We willmeasureadultmortality after 24 h,72h, 7d and 14d,estimatebrood ball formation,andcountemerged adults.Each dose will be replicated 10times with pairs of beetles (480beetles/species).InYear 2, we will identify 4 doses within a narrower rangefor each product on each speciesto more accurately identify the LD50.Pitfalls:Detecting low concentrations of chemical products:Current analytical instrumentation relies on UV absorbance detection, which is subject to interference by unknown co-eluting environmental contaminants. To avoid this, we have budgeted a fluorescence detector (FLD) add-on for our current HPLC system. The FLD is readily compatible with the developed chromatographic method, and will enhance the sensitivity and specificity of the analysis, thereby improving the accuracy of the quantification at lower product concentrations.Collecting and rearing sufficient beetles.While480beetles/doseisa daunting numberto collect eachyear, dung beetles can be quickly collected from fresh cow dung, and are highly reproductive in colonies.We have captured >100On.hecate,and >250Os.rusicolain baited pitfall traps in early summer 2021.Co-PD Kijimoto has keptOn. taurusin large laboratorycoloniesfor15years.Objective 2: We knowlittleabout the community of species inWV.To address this, we propose to survey the community of dung beetles attracted to cow manure in WV with the help of WVU Extension, NRCS andthe WV Conservation Agency(see letters of support;Fig 3).Extensionagentswillsamplethreefarms withthreepitfall traps every month April - October(in total75sites). Beetle collectors will record GPS location of trap, height of surrounding vegetation,canopy cover,andapproximate distanceof nearestcattleto pitfall. PDs will survey producers about their parasite management practices (inconjunctionwith Obj. 3).Samples will be rinsed with water, frozen, and shipped to PD Rowen in Morgantown at regular intervals. Dung beetles will be extracted, identified, and archived in the WVU Arthropod Museum for future work.All beetles from each farm will be pooled across the season to ensure that we have sufficient samplingto estimate total richness.To understand landscape factors that affect dung beetle communities, we will use the USGS cropland data layer and extract information at the 150m and1km scalesto correlate with dung beetle species richness and abundance, focusing on forest and pasture area33,34. In addition, we will use weather data from thenearestNOAAweather station(50+ across the state; https://www.noaa.gov)to estimate 19 Bioclimatic variables (WorldClim.org) for each year of the study.We will also include distance from weather station and elevation in our models.UsingWard's Minimum Variance Clusteringon landscape and bioclimatic variables we will be able to cluster farmswith similar climatesand/orlandscapes35.Usingtheseclusters,we cancompare effects of parasite management on dung beetle communities in different zones.In addition, using partial least squares regression andstructural equation modeling, we canidentify variables that are associated with higher dung beetle abundance and richness36.Objective 3:To increasestakeholder understanding and engagement with dung beetles,first,wewill focus on educatingWVUExtensioncounty agents,and pasture technical professionalson dung beetle ecology and ecosystem services and the known threats to these important organisms. WVU has several outlets for sharing information with county agents, includingAgBeats, a biweekly webinar series hosted every summer, dinner meeting programs,and the Grazing Management School run by Co-PDs Ben Goff and Kevin Shaffer.Second,to reach livestock producers and collect more data,we will establish a dung beetle ID servicecalled "Meet your dung beetles". This will provide producers an opportunity to engage withthe beetles decomposing manure on their own farms, allow them to monitor populations over time, and provide additional beetles for our survey efforts from Obj 2. We will create training videos to show how to set up a pitfall trap using household materials and how to ship dung beetles to us. We will provide a dung beetle report to send back to producers (this includes producers who participate in Objective 2). In addition, information about how to sample dung beetles will be included in field days hosted by WVU Extension, and we will advertise the service through extension newsletters, county offices and at field days, and engage with agricultural families through theagriSTEMCamp in summer 2022 at WVU.Third,we will evaluate the effectiveness oftraining extension personnel about dung beetles andthe dung beetle IDservice.First, we will survey extension county agents about the number of times they have talked about dung beetles in the last year before(Year 1)and after our workshop series(Year 2).In addition,in conjunction with pitfall sampling for dung beetles in Obj 2,wesurvey producersfrom farms that we are samplingon their current parasite control strategies, how they decide to treat, andhow they decide to makeparasite control choices, including dung beetles. We will compare producer answers fromYear 1 andYear 2 as a measure of pre- and post- program effectiveness. Ultimately these surveys will also help usmonitor the important concerns of producers in parasite control in herds over time as our extension programming focuses on IPM and responsive parasite management.

Progress 02/01/23 to 01/31/24

Outputs
Target Audience:Our target audience are cattle producers and extension agents in WV and Entomologists across the US. Changes/Problems:In goal 1 - we changed our approach from a lab-based toxicity assay to a field -based toxicity assay, which is providing more information about many groups of beetles and flies. We were having very poor success in the lab-based toxicity assays with beetle larval survival in control treatments. We have also developed better methods to do these toxicity assays this winter, but do not have enough beetles to complete this objective. Our field-based methods will provide similar information for producers. What opportunities for training and professional development has the project provided?Training of 1 PhD Student, 1 MS Student, and 5 undergraduate students during this reporting period. This has included travel to professional meetings (Eastern Branch Meeting in RI in March 2023, and National ESA meeting in Nov 2023). It has also provided for travel to develop beetle id skills with collaborator Dana Price in MD in summer 2023. How have the results been disseminated to communities of interest?The following presentations and symposium have been organized around this project in this reporting period PD Rowen organized a member symposium at the National meeting in National Harbor MD, titled "Beetles getting it dung: current research in Scarabaeinae and other dung-associated beetles". PD Rowen or students the following oral presentations Brown, H., Lynch, J, Kijimoto, T, Basden, T, Rowen, E.The effect of antiparasitic drugs on selectedScarabeidaespecies in West Virginia.ESA National meeting, National Harbor, VA.(1st place winner) Haridas, S, Lynch, J, Kijimoto, T, Basden, T, Rowen, E. Dung beetle diversity inWest Virginia pastures.ESA National meeting, National Harbor, VA Rowen, E., Basden, T, Shaffer, K, Lynch, J, Kijimoto, T. Meet the dung beetles: Identifying communities and assessing threats to dung beetles in WV.A1261 PD Meeting, Knoxville TN Brown, H., Lynch, J, Kijimoto, T, Basden, T, Rowen, E.The effect of antiparasitic drugs on selectedScarabeidaespecies in West Virginia.ESA Eastern Branch Meeting, Providence RI(2ndplace oral presentation winner Haridas, S, Lynch, J, Kijimoto, T, Basden, T, Rowen, E. Dung beetle diversity inWest Virginia pastures.ESA Eastern Branch Meeting, Providence RI Extension presentations Rowen, E. Digging Deep into Dung Beetles . Mountaineer farm talk. Jan 2024 Rowen, E. Estimating ecosystem services provided by WV dung beetles.Mountaineer Stockman's College.Dec 2023 Rowen, E. Dung Beetle Research on Pasture Health.Grassland Field Day, Guyan Conservation District. Aug 2023 Rowen, E. A guide to WV Dung beetles.WVU AgBeats July 2023 Rowen, E., Basden, T, Shaffer, K, Lynch, J, Kijimoto, T. WV Dung Beetle Survey Update.WV Forage, Livestock, Soil Fertility WorkshopMarch 2023 What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Finish analyzing pesticide concentrations in cow dung using HPLC and will finish collecting arthropod data from field-based toxicity assays by May 2024 - this work will be published in the next reporting period (in M.S. thesis and through two peer reviewed journal publications). Goal 2: We will curate and identify beetles from 23 farms collected in 2023 in the next reporting period. Combined with the beetle data from 2022, this will form the basis for our analysis of beetle communities. We will collate data on weather, location, surrounding landscapes, soils and herd management to better understand the patterns of our beetle communities. In particular, in the next reporting period, we will develop groups of farms given their location, weather and landscape context, and look at the relative effects of intrinsic (landscape etc) versus management on dung beetle communities. Goal 3: We will finish the dung beetle fact sheet by the end of the next reporting period and survey participating extension agents about how they are incorporating dung beetles into their programs after this project.

Impacts
What was accomplished under these goals? Goal 1:In 2023, we developed a field toxicity assay to determine how different products excreted from feces can impact colonization, development, and abundance of different dung beetle species. We homogenized cattle dung dosed with Clarifly (.09, 1, 9µg/g), LongRange (50, 200, 500ng/g), or Safeguard (.3, 3, 10µg/g). Dosed dung was contained in 2-gal buckets that we placed on two farms. We allowed this dung to be colonized for 7 days. Once colonization occurred, we monitored insect emergence in the bins until larvae were no longer detected in the dung. Our analysis investigates the initial colonization of adult insects and the success of the first generation when exposed to increasing doses of anti-parasitic drugs. Data collection to quantify the abundance of dung beetles and flies is on-going. Simultaneously, to assess the exposure of dung beetles to these antiparasitic drugs to understand the risks for dung beetles and the ecosystem services they provide. We quantified the active ingredients using High-Performance Liquid Chromatography (HPLC) with Fluorescent Detection (FLD). to determine the residual concentration of antiparasitic drugs in cattle fecesWe treated heifers with antiparasitic drugs and collected fresh fecal samples periodically for 150 daysin 2023. These samples will be quantified in the next reporting period. Goal 2:In 2023, our collaborators in WVU Extension sampled 23 farms monthly from April-October. These beetles are ready to be sorted, pinned, and identified in our lab. We have also analyzed 2022 data. Specifically, to better evaluate dung beetle community patterns across West Virginia pastures, beta diversity partitioning and indices were used to understand site level and species level contributions to the community. Dung beetle communities in West Virginia pastures show high nestedness across farms, and replacement across seasonal traps. Site level variations could be due to landscape and management factors.Onthophagus hecateandO. taurusare the two species driving variation in communities. Goal 3: In 2023, we engaged with extension and stakeholders through beetle sampling and several presentations on dung beetles throughout the state. We are also in the process of creating a dung beetle fact-sheet for the WVU extension website.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Johnson, L, *Cheshire, L, Rowen, E, Kijimoto, T. Allometric relationships between body width and horn size in the dung beetle Onthophagus hecate (Panzer 1794) (Coleoptera Scarabaeinae). The Coleopterists Bulletin. 77(3): 18. 2023

Progress 02/01/22 to 01/31/23

Outputs
Target Audience:Target audiences reached: · WVU Extension and WV Conservation Agency pasture specialists - we have trained these agents (25) on collecting dung beetles on collaborating farms. · WVU Extension and WVCA havehave reached 36 farmers to talk about and sample dung beetles. · WVU Organic Farm Field Day - reaching organic growers, and community members to educate them about dung beetle ecology · WVU Insect Zoo Halloween event - dung beetle ecology, and demonstration of our dung beetle colonies (~150 people, including local area 2nd & 3rd graders). · Undergraduate/Graduate education - work with dung beetles has contributed to the scientific education of 2 undergraduate students who have been working on pinning, identifying, and creating outreach activities with dung beetles. Work on this project has also contributed to the education of 1 M.S. student, who is learning entomology, toxicology, animal science, and chemistry through her research, and 1 PhD student, who is working on her entomological taxonomy, GIS, and statistical skills through her research with this project.? Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This has included professional development for undergraduate and graduate students, including outreach and research presentations led by students at the WVU Organic Farm Field Day, WVU Halloween at the Insect Zoo and the WV Entomological Society Winter Meeting. How have the results been disseminated to communities of interest?We have reached 36 farmers while sampling their dung beetles, additional organic growers as well as the general public. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will continue to sample dung beetles, recruiting additional farmers to sample, and we will increase outreach activities, extension presentations and publications. In addition, we will conduct dung beetle toxicity assays over the summer during the peak of the dung beetle populations.

Impacts
What was accomplished under these goals? Goal 1: Assess riskofcommon internal and external parasitecontrolmethodstoWV dung beetles To assess the potential exposure of dung beetles in WV to anti-parasite products, thirty animals (n=5) were treated with Eprinomectin, Diflubenzuron, Fenbendazole, Moxidectin, and Ivermectin, with 5 animals left untreated as a control. Fecal samples were taken at 1, 3, 5, 7, 14, 30, 60 & 90 days after treatment from each animal. Methods for extraction of these products from fecal samples has been developed, and graduate student is now quantifying the concentration of each product in frozen fecal samples. To assess the toxicity of these products to dung beetles, four species of dung beetles were trapped and established in colonies in the lab. A slight modification of the timeline for these beetles (move to summer) may be necessary because the colonies have declined in winter, potentially due to natural seasonality for these species. However, we do have a breeding protocol that works during the spring, summer, and fall. Goal 2: Investigate the relationships across WV between dung beetle abundance and richness, landscape, and herd management. This summer, from April - October, our sampling collaborators from WVU extension and WV Conservation Agency fhave sampled from 36 farms and sent beetles. We have pinned >7000 of these beetles, with another 40% of samples left to pin from the summer. A graduate student is working on establishing and streamlining the identification process. We have added 15 new species records from our state to a working species checklist. Goal 3: Educate extension agents and engagestakeholders in dung beetle conservation with a Dung beetle ID service and extension training. We have trained 25 extension agents in how to sample for dung beetles and raised their awareness of dung beetles.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Johnson, L, Cheshire, L, Rowen, E, Kijimoto, T. Allometric relationship between body and horn size in two WV Onthophagus hecate (Panzer) (Coleoptera: Scarabaeinae) populations. Colleopterists Bulletin
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Rowen, E. Fun with Dung: The role of manure in supporting ecosystem services on integrated livestock farms. Oral. ESA, ESC, and ESBC Joint Annual Meeting, Vancouver Canada (Nov 2022)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Haridas, S.& Brown, H. Dung Beetles in Agroecosystems  Diversity and Importance West Virginia Entomological Society Winter Meeting (Jan 2023 )