Progress report for GNC22-358
Project Information
This project will assess the capacity for buckwheat to increase rates of parasitism, and hence death, of house flies, Musca domestica, on dairy facilities in Northern Illinois with the augmentative release of parasitoid wasps. The pupal stage of house flies is attacked by several species of parasitoids. These parasitoids occur naturally and are sold commercially for augmentative releases. Recently, our lab has demonstrated that the house fly parasitoid, Spalangia cameroni experiences greater longevity and nutritional gain from buckwheat, at least in the laboratory (Taylor et al., 2021). Buckwheat is an inexpensive plant and our laboratory results suggest that it may benefit control attempts. The house fly is considered the most important pest of US dairy operations, costing the industry 500 million dollars annually in economic losses (Geden et al., 2021). US dairies make up approximately 25% of the total cash receipts of a $165 billion dollar animal production industry (USDA 2020). An Integrated Pest Management (IPM) approach to the control of house flies is needed because the flies have evolved resistance to insecticides (Freeman et al., 2019), and because insecticides can adversely affect non-target species (Main et al., 2018). Project outcomes include: 1) determine the impact of supplemental buckwheat, placed near where parasitoids are released, on rates of parasitism of house fly pupae and inform our farmer collaborators on parasitoid biological control generally and results of the proposed project specifically; 2) engage with local high school agricultural classrooms on dairy IPM, and capitalize on the students’ social media savvy and connections in creating outreach materials; 3) produce an extension handout of IPM of house flies on dairy facilities and distribute it through Illinois county farm bureaus and participating county extension offices. Buckwheat can improve biological control of crop pests. Whether this success translates to livestock facilities remains to be seen and is what makes this project novel.
Buckwheat borders have increased rates of parasitism and predation by some natural enemies of crop pests (Bianchi et al., 2006). For the proposed project, buckwheat will be placed around dairy cow housing with augmentative release of parasitoid wasps, and parasitoid monitoring stations will be used to gauge rates of parasitism. Each buckwheat plot will be paired with a non-buckwheat plot for comparison. Each plot will have a release of supplemental parasitoids. Pupae from the monitoring stations will be collected and observed for parasitoid emergence to determine rates of parasitism.
This project will involve working directly with dairy producers and a local agricultural class on aspects of house fly control, including how the use of insecticides can lead to resistant populations of house flies and how to implement an IPM program (learning outcomes). From the information provided by this study farmers will be able to institute practices that support natural enemies such as parasitoid wasps (action outcome). At the end of this project farmers and agricultural students are expected to better understand: 1) how resistance to insecticides evolve, 2) impacts of insecticides on non-target arthropods, 3) what parasitoid wasps are, and 4) methods to support those natural enemies of house flies. A local agricultural class will be able to practice science communication skills. They will have the opportunity to be creative in producing educational materials, making use of their social media savvy or producing more traditional materials to garner the attention of other young people or their parents/guardians. The materials generated by the agriculture classroom will be provided to the DeKalb County Farm Bureau. These materials will then be used to create an extension handout with the insight of the next generation of agricultural workers. As a result of this project, farmers will be informed about floral resources as an option for part of their pest management strategies.
Cooperators
Research
Materials and Methods
Flies and Wasps and Buckwheat
The CAR21 strain was used to produce pupae for sentinel stations. Initially, CAR21 pupae were provided by the USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology (CMAVE) in Gainesville, Florida. The CAR21 house fly strain is susceptible to pesticides and has been in colony at the USDA-ARS CMAVE since 2021 (Aldridge et al. 2024). The CAR21 strain of house flies was sourced from a house fly colony that has been in culture since the 1970s. Pupae were placed into a modified 18.9 l plastic bucket. The plastic bucket had a 110 mm diameter access hole, and a 400 mm long fabric access sleeve. A 109 mm diameter and 50 mm long polyvinyl chloride (PVC) pipe was placed inside the fabric sleeve and secured in the bucket access hole. The top of the plastic bucket was covered with a pair of panty hose with the legs tied, secured in place with binder clips. The modified plastic bucket was kept in a 25°C ventilated room at 12:12 h L:D, and the pupae were allowed to eclose. Adult flies were provided with sucrose and tap water ad libitum. At least 24 h before egg collection, flies were also given evaporated milk. Approximately 10 ml of evaporated milk and 10 ml of tap water were mixed and placed with a crumpled piece of paper towel (5 cm x 12 cm) in a 30 ml plastic cup. House fly pupae for experiments were produced by placing an estimated 20,000 house fly eggs in larval media composed of 355 g Calf-Manna, 1500 g of wheat bran, 3750 mL water (Geden et al., 2020).
Spalangia cameroni were received as parasitized pupae from the USDA-ARS CMAVE in Gainesville, Florida, whose S. cameroni colony was a mix of wasps collected from California, Minnesota, Nebraska, and Florida. Parasitized pupae were kept in a cage in a 25ºC incubator at 12:12 h L:D. Buckwheat plants were greenhouse grown from seed (Johnny’s Selected Seeds, Winslow, ME) in two-gallon pots in a mix of 1:1 field loam:potting soil.
Laboratory Experiment
Rates of fly and parasitoid wasp emergence for wasps exposed to fly pupae and buckwheat inflorescences in the laboratory were determined by setting a 1 d.o. S. cameroni in one of two 237 ml glass jars. Both the treatment and control jars contained 30 1-2 d.o. house fly pupae in an open small polystyrene petri dish (30 mm x 15 mm), but the treatment jar also had buckwheat inflorescences and water, whereas the control jar had two waters. The buckwheat inflorescences were cut from the plant and placed into a water filled 2-dram glass vial covered with parafilm (Parafilm™ M Laboratory Film, Heathrow Scientific™, Vernon Hills, IL) to minimize drownings. The parafilm was perforated with a small hole that allowed for placement of a single buckwheat inflorescence. Buckwheat inflorescences were only used if nectar was visible. Water was available by filling a 1.5 ml microcentrifuge with tap water and adding an approximately 30 mm x 5 mm strip of folded KimWipe (KimWipes Delicate Task wipes, Kimberly-Clark Worldwide Inc., Roswell, GA) to prevent drowning. Both the treatment and the control glass jars were covered with fabric that was secured by two rubber bands. Jars were placed into a plastic box (425 mm x 302 mm x 178 mm) and placed into a 25ºC incubator. The parasitoid wasp in each jar was removed after 3 d, and each small petri dish containing the pupae was closed. Then, daily, for 2 weeks any emerged flies and empty puparia were removed from each dish. After 2 weeks the dish was sealed with parafilm, and pupae were checked for S. cameroni emergence for 2 months.
Field Experiment
Ten buckwheat pots per site, with a total of 3 sites, were placed on the dairy farm near areas of ideal filth fly breeding habitat, but out of the way of farm workers. Buckwheat plants were checked daily and watered as needed. A control plot was chosen at least 15 m away from each buckwheat pot and in a similar environment. This distance was chosen as S. cameroni is not known to disperse farther than 5 m away from where released (Machtinger et al., 2015). Rates of fly and parasitoid wasp emergence and diversity of parasitoids on the dairy farm were determined using modified sentinel monitoring described by Geden et al. (2020). Briefly, a sentinel monitoring station consists of a 609 mm x 178 mm x 178 mm wire animal trap (Havahart 1078SR Medium Professional Style 1-Door Humane Catch and Release Animal Trap, Havahart®, Lancaster, PA) containing two 1.18 l plastic food storage boxes (Ziploc brand, SC Johnson, Racine, WI). The trap was placed in the field closed to keep out vertebrates. For each plastic food storage box, powered talc (EZ-slide Talc Powder Seed - Flow Lubricant, Van Sickle Paint, Lincoln, NE) was applied to the rim to prevent ants from entering the box. Each box was filled halfway with larval media in which 3rd to 4th instar house fly larvae were developing. On top of the larval media was placed a sentinel bag made with screen mesh (1.22 m x3 m Durable Fiberglass Screen Mesh, TOOLTRIZ, Jackson, WI). Each sentinel bag contained approximately 100 1-3 d. o. pupae and was closed with a 25 mm metal binder clip. On top of the closed trap was a landscaping stone block (89 mm x 178 mm x 150 mm) to prevent the wind from moving the trap. Geden et al. (2020)’s modified sentinel monitoring has greater sensitivity to the presence of Spalangia spp. compared to the original sentinel bag monitoring technique described by Rutz and Axtell (1980).
Each buckwheat plot and each control plot had three monitoring stations associated with it, with one station at 0 m, one at 1.5 m, and one at 3 m. Sentinel monitoring stations were replaced every 3 d for 4 weeks starting August 20, 2023; and the last station was collected on September 16, 2023, which corresponds to peak parasitoid activity in Northern Illinois (Olbrich and King 2003). Approximately 325 house fly pupae parasitized by S. cameroni were released weekly (August 20 and 27, and September 3 and 9, 2023) at each 0 m monitoring station, taking care that the released parasitized pupae did not enter the sentinel stations.
Fly emergence rate was the percent of pupae in the sentinel bags from which flies emerged. Fly emergence rates were adjusted to account for mortality found in flies not exposed to natural enemies.
For each sentinel station collection, fly pupae were transferred into a 100 mm x 15 mm polystyrene petri dish. Flies were allowed to emerge for two weeks, and adult flies were tallied and removed from the petri dish. The remaining fly pupae were individually placed into a well of a polystyrene 96-well plate (Falcon™ 96-Well, Non-Treated, U-Shaped-Bottom Microplate, Thermo Fisher Scientific, Bartlett, IL) following Geden et al. (2020). Once the pupae from one sentinel station were placed into wells, parafilm was cut to size and used to seal the tops of the wells. On top of the parafilm, a 76 mm x 127 mm notecard was cut to size and placed on top of the parafilm. The lid of the 96-well plate was then placed over the parafilm and the notecard. Masking tape was used to seal the edges of the closed 96-well plate. These measures prevented parasitoid escapes. Parasitoid wasps were allowed to emerge for 3 months following collection of fly sentinel bags from the field. Pupal parasitoids were identified with the Rueda and Axtell (1985b) and Gibson (2000) filth fly parasitoid wasp keys, with identification confirmed independently by three individuals.
Statistics
All analyses except for the log-linked Poisson GLMM were completed using SPSS (IBM Corp., 2019). The log-linked Poisson GLMM was completed using RStudio 2024.12.0+467 (Posit team 2024). In the laboratory experiment, fly emergence was compared when buckwheat was present versus absent, using independent sample t-tests; and the parasitoid wasp emergence was compared using Mann-Whitney U tests because the residuals were not normal. In the field experiment, house fly emergence rate and parasitoid wasp emergence rate were analyzed separately with GLMMs (generalized linear mixed models). The GLMMs used plot (control versus buckwheat) and distance away from the S. cameroni release point (0, 1.5, and 3 m) as fixed factors, with site (1, 2, 3) and collection date (every 3 days, for a total of 9 dates) as random factors. For fly emergence rates, GLMM with an identity link function and a normal distribution was used. Because of a significant interaction for fly and wasp emergence between the fixed effects of treatment and distance (see Results), the effect of each factor was then examined for each value of the other factor. (F5,146 = 13.40, P < 0.001). In other words, the effect of distance was compared for the buckwheat plots and then separately for the control plots, and the difference between the buckwheat and control plots was compared at each distance. Pairwise contrasts were used to determine differences among fixed factor levels when a fixed factor was significant. For parasitoid emergence rates, a log-linked Poisson GLMM was used due to the number of zeros in the data (Perumean-Chaney et al. 2013). Field parasitoid diversity was determined by calculating the Shannon diversity index for each distance within each plot and then comparing between treatments within each distance by Kruskal-Wallis tests.
Results
In the lab experiment, there was no difference between percent fly emergence or parasitoid wasp emergence for S. cameroni exposed to buckwheat and pupae compared to S. cameroni with pupae alone (fly emergence: t38 = 0.33, P = 0.75; mean ± SE: 65.41 ± 3.62; wasp emergence: U10,10 = 229.50, P = 0.43; mean ± SE: 2.00 ± 31.24).
In the field experiment, fly emergence showed a significant interaction between effects of treatment and distance (F2,146 = 13.40, P < 0.001). Fly emergence was greater from buckwheat plots than from control plots at 0 m (F1,42 = 13.40, P = 0.01) but lower for buckwheat plots compared to control plots at 3 m (F1,42 = 31.51, P < 0.001). There was no difference in fly emergence for buckwheat versus control plots at 1.5 m (F1,42 = 2.95, P = 0.09). Fly emergence rates decreased with distance for buckwheat plots (F2,68 = 44.26, P < 0.001), but did not differ with distance for control plots (F2,68 = 0.64, P = 0.53).
For parasitoid emergence there was not a significant interaction between effects of treatment and distance (χ21=1.50, P = 0.22). There was not a significant difference for wasp emergence among the three distances (χ21=2.89, P = 0.09). Parasitoid emergence was over five times greater from buckwheat plots than control plots (estimated marginal mean ± SE: 0.18 ± 0.26 versus 0.03 ± 0.05, χ21=68.27, P < 0.001).
A total of 184 parasitoid wasps emerged from the sentinel pupae spanning five genera and at least seven species (Table 15). The most prevalent species was Muscidifurax raptor followed by Pachycrepoideus vindemmiae. Three species of Spalangia were present: S. cameroni, S. nigroaenea, and S. endius. Two wasps were only able to be identified to the genus level. Two Urolepis spp., both most likely U. rufipes; however, key identifying features were damaged during transport. A single likely member of the Dibrachys cavus complex was also present. There was no difference in the Shannon diversity of parasitoids at buckwheat plots compared to control plots across all sites (F1,136 = 0.04, P = 0.85).
Table 15. The parasitoid wasp species found at a Northern Illinois dairy.
|
Parasitoid Wasp |
Count |
Frequency (%) |
|
Muscidifurax raptor |
108 |
59 |
|
Pachycrepoideus vindemmiae |
52 |
28 |
|
Spalangia cameroni |
13 |
7 |
|
Spalangia nigroaenea |
5 |
3 |
|
Spalangia endius |
3 |
1.5 |
|
Urolepis spp. |
2 |
1 |
|
Dibrachys sp. |
1 |
0.5 |
Discussion
Percent fly emergence and S. cameroni emergence in the laboratory was not significantly affected by the presence of buckwheat inflorescences. In contrast, in the field experiment, relative to the control, the presence of buckwheat appeared to reduce house emergence, but only at 3 m, not 1.5 or 0 m, from where the buckwheat and the S. cameroni release were. Looking just at the buckwheat plots, as distance from buckwheat increased, the amount of fly emergence decreased, whereas in the control plots fly emergence remained consistent at all distances. Based on just the distance from the S. cameroni releases, fly emergence might have been expected to be lowest at the closer distances. But instead fly emergence was lowest at the buckwheat plots at the farthest distance. This may be due to the use of buckwheat by natural enemies of house flies other than S. cameroni that were also present on the dairy. An additional hypothesis for the lower rate of fly emergence for buckwheat plots as distance from buckwheat flowers and the parasitoid release point increased is that the behavior of parasitoids changes around a floral resource. Parasitoids may use the energy gained from the consumption of nectar to disperse rather than increase parasitism efforts near the buckwheat flowers However, why parasitoids would disperse when there were fly pupae near the release site is unclear. Perhaps the increased rate of fly emergence at the release point may be from shading or other protective measures that buckwheat provided fly pupae. Buckwheat is often suggested as a potential supportive measure of natural enemies, and there are studies that support its use as part of integrated pest management strategies (Bianchi et al. 2006, Thurman and Furlong 2024).
The present study suggests that parasitization of house fly pupae is aided by the presence of buckwheat. The percent fly pupae with parasitoid emergence was over five times greater at sentinel monitoring stations in buckwheat plots than in control plots. However, the effect of buckwheat on house fly emergence was more complicated, being distance dependent. In addition to a possible benefit to house fly emergence from shade, adult house flies may also benefit by feeding on buckwheat inflorescences (Grimenstein 2024). White clover, Trifolium repens, is a potential floral source for parasitoids that house flies do not spend a significant amount of time feeding at. However, white clover must be treated with caution due to the risk of dairy cows developing ruminal tympany (Wang et al. 2012). Alternative flowering plants are worth investigating, particularly shorter ones planted where they are unlikely to shade fly pupae, and ones that house fly parasitoids but not house flies feed on, which seems to rule out dandelions and alyssum (Taylor et al. 2022, Grimenstein 2024). Such studies should measure fly emergence not just natural enemy numbers.
Despite the large number of S. cameroni released as part of this study, relatively few S. cameroni, were found from the parasitized pupae. Why is unclear. Potentially, the M. raptor and P. vindemmiae out competed the S. cameroni. Musicidifurax spp. are known for their tendency to outcompete other parasitoids in parasitizing pupae in monitoring set-ups (Floate 1999). The parasitoid species collected from the sentinel pupae somewhat match the parasitoid species collected at another Northern Illinois dairy by Olbrich and King (2003). Both studies found Muscidifurax, S. cameroni, S. endius, and S. nigroaenea from parasitized house fly pupae. In contrast to the present study finding mostly M. raptor and P. vindemmiae, Olbrich and King (2003) found mostly S. nigroaenea.
Additional studies examining the impact of buckwheat on the longevity of Muscidifurax spp. and parasitism rates, including field studies examining the impact of buckwheat combined with supplemental Musicidifurax spp. release. That the present study showed that buckwheat seems to support house fly parasitoids in the field is encouraging, as was the decreased fly emergence 3 m from buckwheat plants. This decreased rate of fly emergence at 3 m suggests that facilities may be able to locate supplemental flowers for parasitoids a few meters away from areas of peak fly activity, which will often be desirable to avoid livestock removing the flowers and to avoid interference with other farm activities.
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Educational & Outreach Activities
Participation Summary:
Currently two outreach/science communication products have been created to provide students with an example of what they can produce as outreach materials for their class assignment.
I gave students an interactive talk on Tuesday March 19th where they will be provided a slideshow presentation, a worksheet, and an assignment related to IPM and natural enemies for filth fly control.
I presented about the evolution of pesticide resistance at STEMfest. STEMfest is a local science, technology, engineering, and math event at Northern Illinois University where STEM fields showcase their fields and what they do members of the STEM community.
This is the in-class note page and assignment for the presentation given to students in an agricultural course.