Progress report for GNC19-280
Bats provide valuable ecosystem services to agricultural crops by pollinating plants such as agave1 and managing pest insects of cotton2. For example, Brazilian free-tailed bats consume enough corn earworm moths each summer to delay the first pesticide application2. These bats’ economic value has been estimated at $22.9 billion per year2. However, the role of bats as a biological control for pest insects in apple orchards has not been fully explored. This project entitled Detection of Tarnished Plantbugs, Apple Maggots, and Codling Moths in Bats’ Diet in Southern Michigan Apple Orchards will focus on analyzing the diet of 4 common bat species in southern Michigan apple orchards. I will determine if they are foraging on 3 common pest insects known to damage apple crops. Bats will be mist-netted in conventional and organic orchards to collect fecal samples. The fecal samples will be analyzed with a genetic technique to identify insects within the fecal sample. In addition, insect samples will be collected to monitor emergence periods. The purpose of analyzing diet is to determine if these bats are foraging on important apple pest insects. Diet results will be compared among orchard management types and bat species to show if the bats are foraging on the pest insects and in what proportion during the insects’ peak emergence periods. This project will emphasize the importance of bats to the agricultural system and provide alternative methods of pest control for farmers to implement into their practices. Furthermore, future implications include estimating the economic value of bats to farmers with regard to pesticide use and pest damage reduction. The outcome of this research is to provide farmers with the knowledge of bat diet and ecology so they may integrate bats as a natural, sustainable, and less expensive alternative to chemical pest control. Bats as a natural pest control method reduce health risks associated with pesticide use and provide an additional source of natural predation. The anticipated outcomes will be evaluated through two surveys each season and two years after to farmers about bat and pest insect abundance on their property and the amount of damage to crops. Participating farmers will also receive a bat house for their property to begin attracting bats to their orchards.
1S. Ducummon, paper presented at the Bat Conservation and Mining Forum, St. Louis, MO, 4-16 November 2000.
2J.G. Boyles et al., Economic importance of bats in agriculture. Science. 332, 6025 (2011).
Farmers will gain a basic understanding of bat ecology and bat diet as it relates to their orchard. Farmers will learn whether bats are foraging on any of the three target pest insects and if they increase predation of these insects during peak emergence periods. This will incentivize farmers in learning how to manage their properties for bats to take advantage of bats’ beneficial pest control properties. Participating farmers will receive a bat house to start attracting bats to their properties for future integration into their pest management program. Farmers who integrate bats may also reduce environmental harm over time with delayed or reduced pesticide applications each growing season.
Outcomes will be measured with surveys sent to farmers at the start and of growing season and for two years post-research. Surveys will include questions regarding an observed estimate of the bat population on their property increasing or decreasing, observed estimate of the pest insect populations on their property increasing or decreasing, and how well they feel bats have contributed to their orchard thus far. I will also ask about their economic gain or loss if bats increased on their property with pesticide applications and pest damage loss. Furthermore, a change in farmers’ behavior will be measured with the survey, addressing any modifications or additions they have made to their property to attract bats more.
During the summer of 2019, bats were trapped in triple-high mist-net arrays deployed in orchard corridors. Mist-nets were concentrated around water, forest edges, and flight corridors1. Mist-nets were deployed from dusk until five hours after dusk. Captured bats were placed individually in new brown paper bags until the bat defecated. Bats were released if they did not defecate within 60 minutes2. Captured bats were marked with a non-toxic marker3 on the fur to identify recaptures. I recorded the capture time, location, species, sex, age class, weight, reproductive status (pregnant, lactating, post-lactating, testes descended, non-reproductive), forearm length, and wing score for a health assessment. Fecal samples were stored with silica gel desiccant beads in the field until placed in a freezer at 4°C until processing.4 Equipment was disinfected using a 1:10 diluted bleach mixture after each use5.
I collected insects from Trécé Pherocon Codling Moth 10x lure, Trécé Pherocon VI Delta traps, and Trécé Pherocon VI Delta liners (Great Lakes IPM, Vestaburg, MI) each netting session. I cleared traps of insects before starting the survey by placing a new liner in the trap. I stored insects from the traps at room temperature in a plastic Ziplock bag or brown paper bag until processing. Insects will be counted and identified to the lowest classification possible to estimate relative abundance each sampling session.
During the summer of 2020 (and continuing through the summer of 2021), bat fecal samples were only collected underneath known roosts because the US Fish and Wildlife Service suspended all unnecessary bat handling for bats’ safety from SARS-CoV-2 (i.e. Covid-19)5. My study area decreased from eight orchards to four orchards, redistributing the orchard type ratio to one organic and three conventional sites. At these four sites, I collected fecal samples from occupied bat houses or man-made structures around the orchards on a plastic tarp under the roost. Any fececes on the tarp between sampling sessions were collected before sampling. The following day, the feces on the tarp were collected again to sample that night’s insect intake. Tarps were set out for a minimum of 1 day and no longer than 28 days. Fecal samples were pooled from the tarp to fill a 15-mL collection tube. Location and time information was also taken. Fecal samples were stored with silica gel desiccant beads and stored in a freezer at 4°C until processing4, and all equipment was disinfected using a 1:10 diluted bleach mixture after each use6.
I also used tanglefoot traps and light traps as insect collection methods. I used the same Trécé Pherocon brand products for the pheromone traps, an 18 oz. red Solo® cup with tanglefoot for the tanglefoot traps, and 3 Trécé Pherocon VI Delta liner surrounding a UV light, lantern, or spotlight for a light trap. Traps were cleared of insects before starting the survey by placing a new liner in the trap, a new cup coated in tanglefoot, or new liners around the lights. Insects from the traps were stored at room temperature in a plastic Ziplock bag or brown paper bag until processing. Insects will be counted and identified to the lowest classification possible to estimate relative abundance each sampling session.
I will use next-generation sequencing (NGS) to identify target insect DNA in the fecal samples. NGS allows for more robust genetic analyses, so I will now be targeting all apple pest orchard insects within southern Michigan apple orchards. Genetic analyses show a higher rate of prey detection compared to taxonomic identification of fragmented insect parts in fecal samples7. I will extract DNA from the fecal samples using a DNeasy Power Lyzer Power Soil kit (QIAGEN, Inc., Valencia, CA) to remove polymerase chain reaction (PCR) inhibitors found in feces. For the 2019 field season, multiple fecal pellets collected from the same bat at the same time will be extracted into a single DNA elution. The roost samples from 2019 and 2020 will be pooled together by date and site, and three replicates per sample will be extracted.
I will use PCR to amplify 150-300 base pairs of the mitochondrial cytochrome oxidase subunit I (COI) region. The amplified DNA will be used in a NGS analysis, where all high-quality insect DNA found in the guano sample will be simultaneously sequenced en masse, yielding millions of raw sequence reads per PCR. Comparison of processed and filtered sequence reads to online databases like Barcode Of Life Database (BOLD) and GenBank will allow me to identify insect DNA, often to the species levels via online sequencing databases like Barcode Of Life Database (BOLD) and GenBank. All molecular techniques will include positive (pure insect samples) and negative (water) controls to account for technical error.
All fecal samples will be scored as having any target pest insect species. The presence of target insects within a bat species’ diet will be summarized as a proportion of all samples collected that night from each bat species. This will allow a comparison between relative insect abundance and ingested insects through the season to understand if more target species are being eaten selectively at times of high local abundance. The relationship between relative target insect abundance and the number of target insects identified in the fecal samples will be analyzed. Relative bat abundance will also be estimated for each orchard. We will explore the role of management strategy (conventional vs. organic) on bat predation of pest insects, but this may not be statistically feasible given the necessary changes to our field sampling strategy in 2020.
1United States Fish and Wildlife Service (USFWS), Indiana bat (Myotis sodalis) draft recovery plan: First revision (2007; https://www.fws.gov/midwest/endangered/mammals/inba/inba_drftrecpln16ap07.html).
2National Park Service (NPS), NPS Institutional Animal Care & Use Committee standard operating procedure for the study of bats in the field (2016; https://www.nps.gov/orgs/1103/upload/NPS-IACUC-Bat-SOP-Final-1.pdf).
3W.L. Gannon, et al., Guidelines of the American Society of Mammalogists for the use of wild mammals in research. J Mammal. 88, 3 (2007).
4A. Corthals, et al., From the field to the lab: Best practices for field preservation of bat specimens for molecular analyses. PLoS ONE. 10, 3 e0118994 (2015).
5U.S. Fish and Wildlife Service, 2020. Agency memo. https://www.fws.gov/southwest/es/Documents/R2ES/AUES_Covid_Bat_Guidance.pdf.
6V. Shelley et al., Evaluation of strategies for the decontamination of equipment for Geomyces destructans, the causative agent of white-nose syndrome (WNS). J Cave Karst Stud. 75, 1 (2013).
7F. Pompanon et al., Who is eating what: Diet assessment using next generation sequencing. Mol Ecol, 21, 1931-1950 (2012).
In summer 2019, I completed 16 trapping nights from 18 July to 12 September, visiting each orchard twice except for Earth First Farm, at which I sampled once. Nine of those nights were successful trapping nights. I captured a total of 60 bats (big brown bat=52, eastern red bat=4, hoary bat=1), and only 3 bats (big brown bat) were recaptures. I collected 65 fecal samples from individual bats (n=59) or roosts (n=6) during the two-week sampling period.
In summer 2020, I collected 320 fecal samples under four known roosts between 19 May and 27 August. I sampled Crane Orchard (conventional) 18 times, Earth First Farm (organic) 17 times, Spicer Orchard (conventional) 15 times, and Erie Orchard (organic)14 times. Samples were collected a minimum of 1 day apart and no more than 27 days apart. I opportunistically completed emergence counts at the roost sites and counted between 10 and 50 bats, with the exception of 1 bat consistently counted at Spicer Orchard.
In 2019, I collected insects on a total of 19 pheromone traps. I placed 1 pheromone trap in most orchards during sampling, but occasionally 2 traps were placed if the netting radius was large. I placed my traps at the start of netting and closed the traps at the end of netting. Traps were not deployed longer than 6 hours. In 2020, I added tanglefoot traps and light traps to my insect sampling. I placed 1 to 2 pheromone and tanglefoot traps at the start of the season. Pheromone and tanglefoot traps were placed for no less than 6 days at a time and no more than 28 days at a time. A total of 50 pheromone traps and 42 tanglefoot traps were collected. I replaced traps each initial collection visit at each orchard. I set out 1 to 2 light traps only during sampling nights, so the traps were placed for 1 to 2 days each visit.
I currently do not have any preliminary genetics analysis or insect abundance results. Covid-19 caused our university to shut down and pushed my timeline back. I am in the process of optimizing my genetics work to send out for NGS.
Educational & Outreach Activities
My outreach involved two presentations for children at Frederik Meijer Gardens and an informational booth at the Downtown Market in Grand Rapids, Michigan. The children’s presentations included a short child-oriented educational session and then a couple of activity stations set up around the children’s garden area where the children, with their parents, could participate in those activities and ask me questions. At the farmer’s market, I set up a booth with children’s take-home activities, fun bat facts for all ages, and a slideshow of bat photos I took during field work. This encouraged passing shoppers as they walked by to stop and ask me what I was doing and why. This opened some great conversation about bat services with apples and other agricultural crops. I spoke to a few vendors, as well, that were interested in how my research could improve their economic gain and other research they could refer to as well.
Since Covid-19 started, some other planned outreach events I had planned were cancelled. I have plans to return to Frederik Meijer Gardens again this summer to do some more outreach with the Children’s Garden and give a presentation to an agricultural audience at Farm Field Day in Hart, Michigan. The anticipated audience will be mostly people in the agricultural industry, with many being fruit farmers. Additionally, I am interested in starting a TikTok or Youtube account to provide free content about bats as a whole, as well as bats in the agricultural field.
I have not gotten the full results of my research yet. I am unable to describe and assess at this point how my project has affected agricultural sustainability or will contribute to future sustainability. I still anticipate that my research will show bats have a positive effect on agricultural sustainability by consuming common apple pest insects. In addition, the change in our laboratory methods to use next-generation DNA sequencing may reveal bat predation on agricultural pest insects beyond the three species we originally proposed to target.
Mine and my advisor’s knowledge, attitude, skills, and/or awareness about sustainable agriculture has not changed. We do still believe bats are a great component of integrated pest management as well as a good source of biological control for organic systems. We have yet to complete the genetic component of my project to see if the fecal samples would prove otherwise.