Final report for GNC24-384
Project Information
Sustainable agriculture is essential to address modern challenges like population growth and resource conservation. Also, it aligns with circular economy principles. The drive towards sustainable agriculture comes from the result of soil erosion, excessive use of pesticides, and other contributing factors. Soil fertility decline is a major concern in all agricultural systems, but particularly in urban settings where farmers are often constrained by contaminants in the soil. A common approach to build soil health is through the applications of amendments. Amendments can provide several benefits, such as improved soil fertility and water holding capacity. Soil amendments can stem from a variety of resources and often are the result of some form of composting. A more recent source of soil amendments are those derived from the production of insects for protein and include Hermetia illucens (black soldier fly; BSF) or Acheta domesticus (house cricket). Recent efforts out of Dr. Ingwell’s lab have focused on BSF because of the advantages associated with their integration on-farm to reduce organic waste and build soil health. The larvae feed on a wide range of organic waste streams and convert those sources into different by-products. Specifically, their use in waste management applications represents a worldwide benefit and the potential to create circular economies. Amending the soil with black soldier fly and other insect by-products can benefit crop production, soil physical properties, and microbial communities. However, knowledge gaps remain around the application rates and impacts on crop production and herbivore susceptibility when insect-derived amendments are utilized. My research aimed to improve our understanding of insect-derived resources as a soil amendment in specialty crop production. Through greenhouse and laboratory assays, I evaluated the impact of three insect-derived soil amendments on susceptibility against two insect herbivores plaguing brassica crop production - Myzus persicae (green peach aphid; GPA) and Trichoplusia ni (cabbage looper). In addition to herbivore performance, I measured plant defenses in the context of total glucosinolate production.
The targeted learning outcomes from this study include 1) a better understanding of the impacts of insect-derived soil amendments on plant defense, 2) urban farmers will improve their knowledge of insect-derived compost benefits in crop production, and 3) farmers increase their ability to utilize insect-derived amendments to improve soil health and crop productivity. Action outcomes from this study will be that urban farmers will be able to 1) improve crop productivity through the application of insect-derived amendments, 2) produce their own insect-derived amendments through black soldier fly composting with locally available organic wastes, and 3) reduce input costs for soil amendments. These outcomes will be achieved through greenhouse research and dissemination of findings at grower and scientific conferences.
Research
Experimental design
The greenhouse experiment was conducted at the Entomology Environmental Laboratory (EEL) greenhouse at Purdue University. The experiment was a randomized complete block design with a total of 10 blocks (replicates). Each block contained a 1-liter plastic nursery pot for every amendment ´ herbivore treatment combination; a single block contained 12 pots. Each pot was filled with a standardized growth substrate and received one bok choy seedling. Plants were exposed to the natural photoperiod during the experimental period (13:11 Day:Night).
Growth substrate preparation and soil amendment treatments
The growth substrate consisted of equal volumes of field-collected soil from Meigs Farm located in Lafayette, IN, sand, BM2® potting media, and the respective soil amendment treatments subsequently described. Field soil was collected from the 5-40 cm depth layer. Insect-derived soil amendments included black soldier fly (BSF) frass and BSF pupal cases, collected from a colony maintained by the Ingwell lab in the EEL greenhouse, and commercial cricket frass (Kickin Frass®, SureSource Agronomy). Unamended substrate served as the control treatment.
To calculate amendment application rates, a composite sample of multiple soil cores from the field at Meigs was collected and sent to A&L Great Lakes Laboratories for analysis using the basic soil package S2 and micronutrient package S3. Additionally, soil samples were analyzed for soil nitrate and ammonium. A sample from each insect-derived amendment source was also sent to the same lab for analysis using the C6 compost analysis package.
The application rates were calculated using the nutrient composition of the field soil and amendments and adjusted to meet the nitrogen (156 kg N/ha), phosphorus (80.7 kg P2O5 /ha), and potassium (89 kg K2O /ha) requirements of bok choy (Table 1). To supplement the insect-derived amendments and reach the plant’s needs, granular conventional fertilizers (46-0-0, 9-23-30) were also added to the amendment-substrate mixtures. Each resulting mixture, containing the insect-derived amendment, substrate, and conventional fertilizer, was bulk mixed and divided among 30 1-liter plastic nursery pots for each treatment combination.
Table 1. Fertilizers and insect-derived amendment application rates applied to bok choy.
|
Application rates (gr/0.0013 sq. ft.) |
|||
|
Amendment (trt) |
46-0-0 |
9-23-30 |
|
|
Control + Fert. |
- |
0.00321 |
0.00414 |
|
Cricket frass + Fert. |
0.09591 |
0.00293 |
- |
|
BSF frass + Fert. |
0.07922 |
0.00307 |
- |
|
BSF pupal cases +Fert. |
0.13830 |
0.00359 |
- |
All pots were irrigated using a drip system installed on the greenhouse benches. Each pot received water through individual 1 GPH emitters, where irrigation frequency and duration were adjusted to meet the plant needs.
BSF amendment sources acquisition
The BSF amendment sources used in this study were BSF frass (insect excrement) and BSF pupal cases (molted exoskeletons or exuviae). Those byproducts, as mentioned earlier, were collected through the rearing process of the BSF colony in the EEL greenhouse. Adult flies were kept in a cage with a bait trap to encourage egg laying. The egg masses were collected three times per week, weighed, and incubated until hatching.
Larvae were then transferred to larger containers and reared on food waste from the Wiley dining hall at Purdue University and organic residues from Meigs horticulture farm. When organic residues were limited, we fed the colony with Gainesville diet which consisted of a combination of wheat bran, alfalfa meal, and cornmeal. The BSF larvae were fed as needed until reaching the prepupal stage, after which they were separated and moved to the insect cage for emergence.
The frass was collected from the rearing bins, weighed, oven-dried at 60°C for 24 hours, and stored in buckets at room temperature until needed. For pupal cases, they were collected after adult emergence, oven dried under the same conditions as the frass and stored similarly.
Plant material
Bok choy seeds (Brassica rapa var. chinensis; Li Ren Choi cv.) were purchased from Urban Farmer (Westfield, IN) and germinated in seedling trays filled with BM2® propagation mix (Berger, US) in a growing room in the Department of Entomology. After developing three true leaves, seedlings were moved to the EEL greenhouse and randomly transplanted into prefilled nursery pots, with one seedling per pot. In total 120 seedlings were transplanted.
Herbivory treatments
Each soil amendment treatment included three herbivory levels: an uninfested control, an infestation with cabbage looper larvae (Trichoplusia ni), and an infestation with green peach aphids (GPA; Myzus persicae). The cabbage looper larvae were purchased from Frontier Agricultural Sciences (Neward, DE) and GPA were obtained from an established colony in the Department of Entomology. A subset of plants remained uninfested and served as the herbivore-free control.
Twelve days after transplanting, the insects were manually placed on bok choy leaves using a fine paintbrush. Within each soil treatment, ten plants were assigned to each of the herbivore treatments. For aphid treatments, five adult GPA were released per plant. For the cabbage looper treatments, one larva was weighed to the nearest 0.0001-g and then released on each plant. After 24 hours, if any of the herbivores were missing or dead, they were replaced. All plants, including herbivore-free controls, were covered with individual perforated bread bags to prevent insect movement between treatments.
Data collection
The data for cabbage looper larvae was collected six days post-infestation. The individual larvae were recovered and weighed using the same laboratory scale as the beginning of the experiment. Then, 14 days post-infestation, all plants were harvested and each plant was cut at the soil surface and a subsample of one gram of fresh tissue was collected from each treatment combination for the glucosinolate analysis. The cabbage looper larvae were removed after 6-days to retain enough plant tissue for the glucosinolate analyses.
For glucosinolate analysis, a total of 36 samples were randomly collected with three samples taken for each treatment combination (amendment x herbivore). The plants were randomly chosen from different blocks to avoid sampling bias. A sample consisted of fresh tissue cut, weighed, and immediately placed into a pre-labeled 2-mL Eppendorf tube. The tube was temporarily stored in an insulated foam container filled with dry ice until the sampling collection was finished. Then, the 36 samples were moved to a laboratory in the Department of Entomology and stored at -80°C. Samples, individually, were taken out from the freezer (-80°C) and were ground using a pestle until it was powdered and immediately moved back to the freezer. Once completed, samples were shipped on dry ice to Creative Proteomics laboratory (Shirley, NY) for quantitative analysis of total glucosinolate content (mmol/g).
The data for GPA populations were collected 14 days post-infestation, on the same day of the harvesting process. The total number of individuals were counted on every plant using a stereo microscope. All the remaining plant material from all treatments was weighed, placed in individual paper bags, and oven-dried (50°C) for 24 hours.
Data analysis
All experimental data was analyzed using JMP 16.1 software (SAS, Inc, Cary, NC). The homogeneity of variance and normal distribution were confirmed and in cases of non-normal distribution, data were log-transformed prior to the analysis. A two-way analysis of variance (ANOVA) was conducted to evaluate the differences in soil treatments, herbivore treatments, and soil ´ herbivore treatment combinations on dry plant weight and total glucosinolate content, individually. Moreover, linear mixed models were used to evaluate the GPA population and cabbage looper larval weight, individually. For both statistical models, block was included as a random factor and statistical significance was determined at a = 0.05. Tukey tests were used to confirm significance among the dependent variables.
BSF byproduct collection through the BSF rearing process and nutritional analysis
The byproducts collected through the BSF rearing process include BSF frass and BSF pupal cases. These materials were generated as part of colony maintenance. Figure 1 illustrates the stage at which the byproducts were separated for use in this study.
Following collection, the insect-derived materials (including cricket frass, which is commercially available) were sent for laboratory analysis to assess their nutritional content. Table 2 indicates the results of the nutrient-rich organic materials, highlighting the elevated nitrogen content (TN) and soluble salts, and a high percentage of organic matter. The high organic matter suggests potential benefits for soil structure and microbial activity (Diacono and Montemurro 2010). On the other hand, the high levels of soluble salts highlight the importance of considering the crop sensitivity to salt and application rates to avoid potential damage.
Table 2. Nutrient composition of insect-derived amendments received from A&L Great Lakes Laboratory via the C6 compost analysis procedure.
|
Nutrient/Soil Characteristic |
BSF frass |
BSF pupal cases |
Cricket frass |
|
OM (%) |
81.19 |
82.44 |
70.36 |
|
pH |
6.6 |
6.7 |
5.6 |
|
TN (ppm) |
36600 |
79500 |
35800 |
|
P (ppm) |
13000 |
7500 |
21600 |
|
P2O5 (ppm) |
30000 |
17200 |
49700 |
|
K (ppm) |
21700 |
14300 |
13900 |
|
K2O (ppm) |
26000 |
17100 |
16700 |
|
Mg (ppm) |
4800 |
2900 |
3500 |
|
Ca (ppm) |
8200 |
18000 |
43900 |
|
Na (ppm) |
11900 |
12000 |
2800 |
|
S (ppm) |
4700 |
3700 |
3900 |
|
Zn (ppm) |
101 |
65 |
182 |
|
Mn (ppm) |
94 |
149 |
222 |
|
Fe (ppm) |
15000 |
100 |
1300 |
|
Cu (ppm) |
14 |
7 |
169 |
|
Al (ppm) (ppm) |
1000 |
200 |
800 |
|
Soluble Salts (1:2) (mmhos/cm) |
11.38 |
12.76 |
8.18 |
OM = organic matter, TN = total nitrogen, P = phosphorus, P2O5 = phosphate, K = potassium, K2O = Potash, Mg = magnesium, Ca = calcium, Na = sodium, S = sulfur, Zn = zinc, Mn = manganese, Fe = iron, Cu = copper, Al = aluminum.
Oven-dried plant weight
The soil amendment x herbivore combination impacted the average oven-dried plant weight (F6,99 = 2.68, p = 0.02). This suggests that the composition of the insect-derived amendments and the herbivore identity determine how well these amendments affect plant performance. Moreover, these organic sources may influence plant development and stress tolerance mechanisms.
Figure 2 shows that plants amended with BSF frass and cricket frass had a comparable oven-dried plant weight to the control treatment, regardless of whether they were infested with an herbivore or not. A similar result happened with plants amended with BSF pupal cases and exposed to GPA. This suggests that plants amended with the insect-derived amendments increased their tolerance to herbivore attack, as reflected in similar plant weight to the herbivore-free control within the soil treatment. These findings align with a previous study by Barragán-Fonseca et al. (2023), which showed that BSF exuviae (pupal cases) can improve plant tolerance to insect attack by sustaining growth and seed production.
On the other hand, plants amended with BSF pupal cases and exposed to cabbage looper larvae exhibited the lowest weight (0.20 g) among all treatment combinations and showed a significantly lower weight compared to the herbivore-free control within the same soil treatment. This result suggests that plants amended with BSF pupal cases are more palatable to cabbage loopers, compared to the untreated control or those amended with BSF frass, but they did not differ from cricket frass. This could be the result of nutritional differences among the plants or a plant defense mechanism that is inhibited by pupal case amendments.
Herbivore infestation on plants
We confirmed with the statistical analyses that neither the cabbage looper larvae weight after six days of infestation on bok choy (F2,8.6 = 2.62, p = 0.13) nor GPA populations after 14 days of infestation (F3,26.4 = 0.79, p = 0.51) were impacted by the soil treatments. These results suggest that the three insect-derived amendments did not directly influence herbivore resistance under the conditions tested. However, the larvae recovery was different between treatments. In this study, the BSF frass treatment had the lowest larvae recovered (four larvae), contrary to cricket frass (eight), BSF pupal cases (seven), and control (seven) soil treatments.
The absence of significant herbivore results aligns with a previous finding by Barragán-Fonseca et al. (2023), which found no early significant decrease in the number of aphids or caterpillars on plants amended with insect exuviae (pupal cases). On the contrary, Wantulla et al. (2024) reported that the effects of Delia radicum (cabbage root fly) performance are influenced by the application rates of such amendments. They found that Brussel sprout plants amended with BSF frass at a rate of 1 or 2 g/kg did not have a significant effect on D. radicum performance, whereas higher application rates (5 g/kg) had a decrease in larval biomass and survival.
In this study, the amendment rates applied were relatively small (Table 1) compared to those in previous studies, which highlights the importance of the dose-dependent dynamics and suggests that evaluating higher rates than those used in this study may be necessary to find effects on herbivore performance.
Total glucosinolate content in plants
The total glucosinolate content in leaves was determined to evaluate how the different insect-derived amendments and herbivores trigger these secondary chemical defense compounds. The findings of this experiment indicated that the soil amendment x herbivore combination significantly impacted the total glucosinolate content (F6,22.28 = 15.0, p < 0.0001).
The results across the herbivore-free controls indicated that plants grown in unamended soil had the highest total glucosinolate content (1.97 mmol/g) compared to plants amended with insect-derived resources. Thus, suggesting that insect-derived amendments may alter constitutive defense levels, while unamended herbivore-free plants maintain a higher constitutive level. Although the insect-derived amendments were applied to meet the nitrogen (156 kg N/ha), phosphorus (80.7 kg P2O5 /ha), and potassium (89 kg K2O /ha) requirements of bok choy, the availability of nutrients and other chemical characteristics (e.g., pH, C/N ratio, and soluble salts) among the soil treatments may have influenced glucosinolate synthesis.
At the same time, herbivory had a soil-dependent effect on the content of total glucosinolate. In cricket frass amended soils, the cabbage looper treatment had a higher glucosinolate content (2.70 mmol/g) compared to GPA and herbivore-free plants. In contrast, similar significant increases in total glucosinolates were not consistently observed in the BSF frass, BSF pupal cases, or unamended soil treatments. These differences may be partially explained by variation in visible feeding damage among the plants sampled, as differences in herbivory intensity can influence the induction of total glucosinolates.
GPA infestations on plants amended with BSF pupal cases also resulted in a significant increase in the total glucosinolate content (2.09 mmol/g) compared to cabbage looper feeding and herbivore-free plants within the same soil treatment.
The glucosinolate results observed in this study suggest that the interaction of insect-derived amendments and herbivory can influence plant defense responses. However, the level of these effects varied depending on amendment composition, nutrient availability, and herbivore presence. Therefore, this is not a one-size-fits-all solution to managing insect pests but has the potential to be developed in a way to maximize plant protection and productivity.
References
Barragán-Fonseca, Katherine Y., Liana O. Greenberg, Gerrit Gort, Marcel Dicke, and Joop J. A. van Loon. 2023. “Amending Soil with Insect Exuviae Improves Herbivore Tolerance, Pollinator Attraction and Seed Yield of Brassica Nigra Plants.” Agriculture, Ecosystems & Environment 342 (February): 1–9. https://doi.org/10.1016/j.agee.2022.108219.
Diacono, Mariangela, and Francesco Montemurro. 2010. “Long-Term Effects of Organic Amendments on Soil Fertility. A Review.” Agronomy for Sustainable Development 30 (2): 401–22. https://doi.org/10.1051/agro/2009040.
Wantulla, Max, Marcel Dicke, and Joop J. A. van Loon. 2024. “Effects of Amending Soil with Black Soldier Fly Frass on Survival and Growth of the Cabbage Root Fly (Delia Radicum) Depend on Soil Type.” Journal of Pest Science 97 (3): 1451–59. https://doi.org/10.1007/s10340-023-01710-9.
Educational & Outreach Activities
Participation summary:
In 2025, I disseminated my research findings through extension and academic venues, with the goal of increasing the understanding and adoption of insect-derived byproducts, especially black soldier fly, on specialty crop production. These activities allowed me to engage diverse audiences, including farmers, undergraduate students, graduate students, and researchers. These also allowed me to communicate the relevance of insect-derived amendments.
In January 2025, I presented a poster at the Horticultural Conference and Expo in Danville, Indiana, where I shared results on the use of three insect-derived byproducts as amendments in specialty crop production. This conference provided opportunities to interact directly with farmers, as well as conversations with extension educators, faculty, and students. There were 150 attendees at this conference.
In March 2025, I presented a poster at the Indiana Small Farm Conference in Danville, Indiana. The audience consisted of farmers, faculty, and students and there were 246 attendees. These interactions facilitated the sharing of my research findings and helped participants translate its application into practical insights.
In September 2025, I had the opportunity to attend the Black Soldier Fly Conference in Cambridge, United Kingdom. I presented a poster on the potential of three insect-derived soil amendments on plant total glucosinolate content. This international conference provided an opportunity to share the results and engage in discussions on innovation, scalability, and future research needs related to black soldier flies. The conference is a small gathering for intimate conversations and networking; there were around 75 attendees from around the world.
In November 2025, I gave an oral presentation at the Entomological Society of America Annual Conference in Portland, Oregon. This conference gave me the opportunity to share my findings with a scientific audience, highlighting insights into how insect-derived amendments influence plant glucosinolate production. The participation in this conference also helped advance discussions on black soldier fly research and strengthened interdisciplinary connections.
Project Outcomes
This project contributed to increasing the visibility of insect-derived amendments, particularly black soldier fly byproducts, in academic and extension-focused venues. The conferences created opportunities to share the findings and introduce the impacts of insect-derived amendments to diverse audiences, including farmers, students, and researchers. The interactions during those conferences showed curiosity in the practical aspects of BSF systems and insect-derived amendments applications. Several farmers showed willingness to establish a BSF colony on their farms or households, while others mentioned already maintaining composting areas with BSF. Attendees actively ask for information about colony establishment, such as the source of larvae and how to make low-cost baited traps. These interactions suggest that the project improved technical understanding and motivated interest in applying it, as well as increased confidence in the feasibility of these practices. It also places insect-derived amendments as potential tools for improving soil health, crop productivity, reducing reliance on conventional inputs (e.g., fertilizers and commercial compost), and enhancing plant tolerance to insect attack.
During the course of this project, I gained valuable experience working with black soldier flies and their byproducts, while further developing skills in project management and scientific communication for diverse audiences. This project provided valuable experience in greenhouse research, which included experimental design, setup, and solving technical problems.
Additionally, I worked with different food waste streams, converting them into valuable resources for agricultural applications. This experience strengthened my understanding of waste conversion and the role of insect-derived materials in different research contexts. Through this work, I developed stronger technical and analytical skills.
Although the insect results did not fully match our expectations, the project generated interesting results that may help improve plant tolerance and resistance to insect attack. These findings provided important insights and highlighted new directions for future study.
This project strengthened my skills as a researcher, which enhanced my analytical thinking, problem-solving abilities, and capacity to communicate results effectively to a relevant audience. Furthermore, this project created meaningful connections with researchers and farmers, supporting knowledge exchange and encouraging the implementation of black soldier fly applications in agricultural systems. It also contributed to a broader awareness of sustainable practices and the importance of addressing current environmental challenges.