Feb 10, 2026
Toxicity Testing of Compounds in Galleria mellonella Larvae
- Juan Jose Quispe Haro1
- 1University of Helsinki
- Galleria protocols
Protocol Citation: Juan Jose Quispe Haro 2026. Toxicity Testing of Compounds in Galleria mellonella Larvae. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1k7wkgr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: February 10, 2026
Last Modified: February 10, 2026
Protocol Integer ID: 242969
Keywords: Galleria mellonella, toxicity testing, invertebrate model, drug screening, larval injection, acute toxicity, survival assay, galleria mellonella larvae this protocol, galleria mellonella larvae, using galleria mellonella, toxicity testing of compound, preliminary toxicity screening, method for toxicity screening, cost preliminary toxicity screening in an invertebrate model, toxicity screening, galleria mellonella, larvae per treatment group, larvae, test compound, microbial metabolite, assay, 7th instar larvae, test compounds via the last proleg, chemical compound, compound, nanoparticle
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Abstract
This protocol describes a method for toxicity screening of chemical compounds, nanoparticles, or microbial metabolites using Galleria mellonella larvae. The procedure utilizes 7th instar larvae injected with test compounds via the last proleg, followed by survival monitoring over 72 hours. With a sample size of 10+ larvae per treatment group, this assay allows for statistically strong, rapid, low-cost preliminary toxicity screening in an invertebrate model that shares key physiological similarities with mammals.
Guidelines
- Dose volume: Do not exceed 10–20 µl per larva to avoid mechanical damage.
- Vehicle toxicity: Always include vehicle control; DMSO >10% can be toxic.
- Larvae quality: Use only healthy, active larvae of similar size.
- Sterility: Work aseptically to avoid infection-related death.
- Replicates: Repeat experiment at least 3 times independently for statistical power (total n=30 per group across replicates).
Materials
Galleria mellonella larvae: 7th instar larvae (≈300 mg each, cream-colored, active). Minimum 10 larvae per treatment group + 10% extra for selection.
Hamilton syringe (10 µl) with removable needle.
6-well cell culture plates (sterile, non-treated).
Optional: Dissecting microscope or magnifying lamp.
Troubleshooting
Safety warnings
Follow institutional guidelines for invertebrate use. Euthanize larvae by freezing at -20°C for 2 h after experiment.
Before starting
Acclimate larvae at experimental temperature for 24 h before injection.30 °C or experimental temp.
Prepare compound solutions freshly on the day of injection. Filter-sterilize if possible (0.22 µm).
Plan groups (n=10-16 larvae per group):
Group 0: Optional handling control (to test if larvae survive handling/physical trauma).
Group 1: Vehicle control (same solvent used to prepare the compound solutions).
Group 2: Positive control (substance known to be toxic with similar mode of action of test compound)
Groups 3-X: Test compounds at desired concentrations (e.g., low, medium, high dose, or serial dilutions).
Assign random 7th instar larvae to each group to avoid health/size bias.
Injection Procedure
Surface sterilize each larvae before injection by wiping each larva gently with 70% ethanol and allowing to dry on filter paper.
Prepare a Hamilton syringe with 10 µl of the desired solution.
Grab a larva by its sides with the thumb and the index fingers and flip it upside down to expose its abdomen.
Localize the hind-left proleg of the larva and carefully insert the needle of the syringe about 5 mm into the body of the larva.
Inject the contents of the syringe slowly to prevent any leaking, and remove the syringe.
Place each individual larva in a well of a 6 or 12-well dish and repeat for all the larvae and conditions.
Incubation and Monitoring
Transfer the dishes to an incubator and incubate the larvae for 72+ hours at desired temperature with 12-12 hours illumination cycle. Do not provide food during experiment.
Monitor survival at 0, 2, 4, 6, 8, 12, 24, 48, 72 hours post-injection (adjust based on kinetics).
Monitor health using the Health Score Index points system every 24 hours.
Modified from DOI: 10.1080/21505594.2021.1950269
| A | B | C | |
| Category | Description | Score | |
| Activity | no movement | 0 | |
| movement only after stimulation | 1 | ||
| letargic movement | 2 | ||
| whole body movement | 3 | ||
| Cocoon formation | no cocoon | 0 | |
| partial cocoon | 1 | ||
| full cocoon | 2 | ||
| Melanization | black larvae | 0 | |
| black spots on brown larvae | 1 | ||
| ≥3 spots on beige larvae | 2 | ||
| <3 spots on beige larvae | 3 | ||
| no melanization | 4 |
Data analysis
Calculate percent survival per group over time and plot Kaplan–Meier survival curves. This can be done easily in GraphPad Prism.
Compare groups using log-rank test or Cox proportional hazards (if multiple doses).
Plot the average health score vs time. Analyse the data by performing a Repeated Measures ANOVA, and post-hoc tests (Bonferroni or Tukey's HSD) to determine if the differences are significant.
Determine the LD₅₀ using the following formula:
where:
xₖ = Lowest concentration that produces 100% mortality
d = Log-interval between doses (10-fold dilutions)
Σpᵢ = Sum of the proportions of mortality for all doses up to but not including xₖ
Protocol references
Larvae Health Index Scoring System, modified from Establishing an invertebrate Galleria mellonella greater wax moth larval model of Neisseria gonorrhoeae infection, Virulence, 2021 Jul 25;12(1):1900–1920. doi: 10.1080/21505594.2021.1950269