Grower-Based In Vivo Propagation of Entomopathogenic Nematodes
January 9th, 2026
We present two methods for farmers to mass-propagate entomopathogenic nematodes as bio-insecticides. The first method uses polyacrylamide gel as the nematode culturing substrate, while the second uses polymer-coated cotton wool as the culturing substrate. Both methods rely on in vivo propagation of nematodes within insect cadavers.
Our research develops a sustainable, grower-friendly method for producing entomopathogenic nematodes, addressing cost and technical barriers to enable on-farm pest control solutions. This low-cost and low-input approach for entomopathogenic nematode culturing leads to sustainable production and is easily adoptable to various nematode species. To begin the initial inoculum, obtain the desired entomopathogenic nematode species.
Use the infective juvenile stage of Steinernema carpocapsae ALL strain and Heterorhabditis bacteriophora VS strain. For individual infections, inoculate 50 live Galleria mellonella larvae with infective juveniles at a density of 100 infective juveniles per insect. Keep the inoculated insects at 25 degrees Celsius until infective juveniles are near emergence.
For mass inoculation, place one infected cadaver with 50 live Galleria mellonella larvae into a plastic container with a volume between 5.7 and 14.2 liters, lined with a moist paper towel and keep the container at 25 degrees Celsius. After 72 hours, remove cadavers that do not show appropriate color changes to avoid contamination. Identify tan or brown cadavers for Steinernema infections, and red or orange cadavers for Heterorhabditis infections.
Next, moisten the polyacrylamide gel with water at a ratio of 20 milliliters per dry gram of gel. Place the cadavers on top of a nylon screen mesh with a 2-by-2 millimeter pore size, and transfer the appropriately colored cadavers into the clear plastic container containing polyacrylamide gel. Then remove and discard depleted cadavers once infective juvenile production diminishes substantially.
Harvest infective juveniles from the infected hosts that remain on the screen above the gel. Continue harvesting into the gel for five to seven days after infective juveniles begin to emerge. To develop inoculum for subsequent production rounds, expose healthy Galleria mellonella larvae to infective juveniles within the gel in 90-millimeter Petri dishes.
Use these newly infected hosts to inoculate the next group of Galleria mellonella larvae. For entomopathogenic nematode applications, rinse infective juveniles that have entered the gel using tap water and screens with a pore size smaller than 0.3 centimeters. Alternatively, apply the nematodes directly in the gel to small arenas without rinsing.
To obtain infective juveniles of Oscheius onirici and Heterorhabditis georgiana from in vivo nematode cultures with Tenebrio molitor as the host, gently irrigate the round cotton pad with water. Position four to six Tenebrio molitor larvae, either live or freeze-killed, in a non-overlapping pattern between the two cotton pads and add 10 to 15 milliliters of water. Add 10 milliliters of a nematode slurry with a concentration of 400 to 500 infective juveniles per milliliter onto the top cotton pad, and allow the infective juveniles to infect the mealworms.
Harvest infective juveniles from the Petri dishes by pouring 20 to 25 milliliters of water over the top cotton pad, then collect the slurry that is percolated through the cotton. To infect insect host in large shallow trays, cut and place the wool bilayer within the tray. Then distribute the Tenebrio molitor hosts between the wool sheets.
Before thoroughly drenching the wool sheets with approximately 400 milliliters of a nematode suspension at a concentration of 80, 000 to 90, 000 nematodes per liter. Incubate the tray at 21 to 22 degrees Celsius for four weeks. After four weeks, harvest the nematodes by irrigating each tray with 500 milliliters of tap water and then collecting the solution.
Repeat the irrigation a second time and collect a total of one liter of nematode slurry. Finally, determine the nematode density by counting the number of nematodes per milliliter under a dissecting microscope and tally the counts for each replicate tray. In the Steinernema carpocapsae experiment, setting 30 cadavers in polyacrylamide gel in 14.2-liter clear plastic containers produce significantly more infective juvenile nematodes per cadaver compared to that in larger white traps.
In the Heterorhabditis bacteriophora experiment, significantly more infective juvenile nematodes per cadaver were produced in the grower-oriented gel treatment compared to the white trap treatment. Grower-oriented EPN production methods represent lower cost, adoptability, sustainability, longevity, and higher yields for pest control programs. EPNs are suitable for a wide diversity of cropping systems.
Unlike prior methods, our grower-based approaches, once initiated, are fully self-sustaining. Medium gel can be stored far months, under refrigeration, for later use, making it cost effective. This research provides a clear path for farmers and stakeholders to propagate EPNs in small areas without much technical difficulty.
In the future, further optimization and increases in efficiency can be explored.
Overview
This article presents two grower-oriented methods for mass-propagating entomopathogenic nematodes (EPNs) as bio-insecticides. The first method compares the standard White trap technique with a polyacrylamide gel-based substrate for in vivo production of Steinernema carpocapsae and Heterorhabditis bacteriophora, showing improved nematode yields. The second method uses live Tenebrio molitor larvae embedded in polymer-coated cotton wool to propagate native species Oscheius onirici and Heterorhabditis georgiana, with nematodes harvested via systematic irrigation to create a field-ready slurry. Both approaches aim to provide farmers with simple, scalable, and independent nematode production systems.
Key Study Components
Area of Science
- Entomology
- Biological pest control
- Nematode mass production
Background
- EPNs are effective biocontrol agents for insect pests.
- In vivo propagation yields high-quality nematodes but is labor-intensive and costly.
- In vitro methods are more cost-effective but may produce lower-quality nematodes.
- Existing grower systems rely on external labs for inoculum calibration, reducing reliability.
Purpose of Study
- To develop reliable, grower-based EPN propagation systems.
- To enable farmers to produce their own nematodes after initial inoculum acquisition.
- To improve accessibility and scalability of EPN use in agriculture.
Methods Used
- In vivo nematode production using White trap method.
- Grower-oriented in vivo production using polyacrylamide gel as substrate.
- Mass propagation of native EPNs in Tenebrio molitor larvae.
- Embedding larvae between polymer-coated cotton wool layers.
- Systematic tray irrigation to harvest emerged nematodes into a slurry.
Main Results
- Polyacrylamide gel method produced more nematodes than White trap for S. carpocapsae and H. bacteriophora.
- Native species O. onirici and H. georgiana were successfully propagated in T. molitor larvae.
- Nematodes were efficiently harvested via irrigation, forming a field-applicable slurry.
- Both methods are technically simple and scalable for grower use.
Conclusions
- Grower-oriented systems reduce reliance on external laboratories.
- Methods enable continuous, on-farm EPN production.
- Approaches support greater farmer independence and profitability.
- Systems have potential for wide adoption in sustainable pest management.
Frequently Asked Questions
Executive Industry Relevance
For biopharma R&D, scalable and reliable production of biological agents is critical for target validation and assay development. This article demonstrates grower-oriented systems for mass-producing entomopathogenic nematodes, offering a model for decentralized, reproducible bioproduction that reduces dependency on centralized facilities. Such approaches support mechanistic de-risking in early discovery by enabling consistent supply of bioactive agents for phenotypic screening and translational biomarker studies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses through reliable production of nematode-based bioactive agents for functional target validation.
- Operational Value: Supports biological de-risking by providing scalable access to high-quality entomopathogenic nematodes for mechanism-of-action studies.
- Predictive Value: Facilitates portfolio triage by allowing reproducible generation of pest control agents to assess target engagement and efficacy in disease-relevant systems.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream workflows by ensuring consistent nematode output for assay standardization.
- Operational Value: Addresses assay reproducibility and quantitative outputs through systematic irrigation harvesting, enabling reliable compound evaluation in screening campaigns.
- Scalability: Highlights platform reuse and scalability of grower-oriented systems for high-throughput phenotypic screening applications.
Translational & Preclinical Research
- Scientific Value: Discusses disease relevance and translational biomarker alignment through use of native nematode species in live insect hosts, mimicking natural host-pathogen interactions.
- Operational Value: Describes continuity from discovery through preclinical validation by enabling on-farm production that mirrors industrial bioproduction scalability.
- Risk-Adjusted Advancement: Addresses risk-adjusted advancement decisions by reducing variability in agent supply, supporting consistent preclinical dosing and efficacy testing.
Pipeline & Workflow Integration
Positioned within the discovery continuum, this method supports early discovery through lead identification by providing a reproducible source of nematodes for target validation and assay development, with potential extension into preclinical work when supported by source material.
- Discovery Biology: Explains how the method supports hypothesis testing, pathway clarification, and biological de-risking through reliable production of entomopathogenic nematodes for functional studies.
- Screening: Describes assay readiness, reproducibility, and quantitative outputs via nematode slurry generation, supporting reliable compound screening and hit validation.
- Analytics: Highlights measurements such as nematode yield and harvest efficiency that enable teams to compare production conditions and assess process robustness.
- Translational Research: Connects the method to preclinical continuity through use of native species in live hosts, supporting biomarker alignment and disease-relevant system modeling.
- Enterprise Reuse: Frames the method as a reusable capability rather than a single-use technique, enabling decentralized bioproduction across research sites or collaborative networks.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence, target validation, and reduction of mechanistic ambiguity through consistent agent supply.
- Operational Value: Standardization, reproducibility, and scalability of nematode production via simple, grower-friendly systems.
- Strategic Value: Better go/no-go decisions, capital efficiency, and reduced late-stage biological risk by ensuring reliable access to bioactive agents.
- Portfolio Impact: Risk-adjusted prioritization and advancement decisions supported by reduced variability in agent production and quality.
Implementation Considerations
- Required scientific expertise in nematode culture and insect host management.
- Instrumentation and analytical infrastructure needs include tray irrigation systems and microscopy for nematode quantification.
- Cross-team standardization requirements for inoculum sourcing and propagation protocols across sites.
- Adaptation considerations across model systems, including alternative insect hosts and nematode species.
- Practical limitations include dependency on initial inoculum from established laboratories and environmental controls for optimal yield.
