PiggyBac-mediated transgenesis and CRISPR/Cas9 knockout in the greater wax moth, Galleria mellonella
Pearce JC, et al. Lab Animal (Feb 2026) — DOI: 10.1038/s41684-025-01665-7
Why this matters
This study reports the first successful genetic engineering toolkit in Galleria mellonella — combining PiggyBac transgenesis and CRISPR/Cas9 gene editing to generate genetically modified wax moth lines. Until now, Galleria has been widely used in infection and immune research, but lacked reliable tools to modify its genome directly. This work overcomes that limitation, enabling researchers to insert, knock out, or modify specific genes in Galleria for the first time.
The GMRC continues to develop this technology, with the aim of generating additional transgenic and CRISPR-edited Galleria lines to help dissect host infection responses in this valuable partial-replacement model organism.
Pearce JC, et al. Lab Animal (Feb 2026) — DOI: 10.1038/s41684-025-01665-7
Why this matters
This study reports the first successful genetic engineering toolkit in Galleria mellonella — combining PiggyBac transgenesis and CRISPR/Cas9 gene editing to generate genetically modified wax moth lines. Until now, Galleria has been widely used in infection and immune research, but lacked reliable tools to modify its genome directly. This work overcomes that limitation, enabling researchers to insert, knock out, or modify specific genes in Galleria for the first time.
- These methods make Galleria a genetically tractable model organism, expanding its use beyond descriptive infection studies into functional genetics.
- Engineered lines can be designed to carry reporters or functional knockouts, improving real-time analysis of host–pathogen interactions and responses to antimicrobial agents.
- Because Galleria can be maintained at human-relevant temperatures and mirrors key aspects of mammalian innate immunity, this genetic platform offers a scalable, ethical alternative to rodent models for early-stage infection and antimicrobial research.
The GMRC continues to develop this technology, with the aim of generating additional transgenic and CRISPR-edited Galleria lines to help dissect host infection responses in this valuable partial-replacement model organism.
Cold-Shock-Mediated Inhibition of Silk Extrusion in Galleria mellonella
Matiya DJ, Tutt K, Wakefield JG, Campbell JS. bioRxiv (Jan 2026) — DOI: 10.64898/2026.01.20.700541
Why this matters
This preprint describes a simple cold-shock method that reliably suppresses silk extrusion in Galleria mellonella larvae, improving ease of handling during injections and other experimental manipulations. Silk production during handling can complicate infection studies by interfering with positioning and survival assessment; this method addresses that challenge by transiently inhibiting silk extrusion without compromising larval viability.
In the study, larvae exposed to short cold-shock at −20 °C completely suppressed silk extrusion while maintaining 100 % survival. The approach is straightforward to implement and reproducible, offering a clear procedural advantage over earlier chilling techniques.
While cold-shocked larvae showed some developmental delay and changes in body weight and fecundity, they remained suitable for use in infection experiments, responding comparably to controls when challenged with Escherichia coli.
By enhancing handling and experimental safety without altering pathogen susceptibility, this method provides a practical refinement for researchers using Galleria mellonella as a host model in infection biology.
Matiya DJ, Tutt K, Wakefield JG, Campbell JS. bioRxiv (Jan 2026) — DOI: 10.64898/2026.01.20.700541
Why this matters
This preprint describes a simple cold-shock method that reliably suppresses silk extrusion in Galleria mellonella larvae, improving ease of handling during injections and other experimental manipulations. Silk production during handling can complicate infection studies by interfering with positioning and survival assessment; this method addresses that challenge by transiently inhibiting silk extrusion without compromising larval viability.
In the study, larvae exposed to short cold-shock at −20 °C completely suppressed silk extrusion while maintaining 100 % survival. The approach is straightforward to implement and reproducible, offering a clear procedural advantage over earlier chilling techniques.
While cold-shocked larvae showed some developmental delay and changes in body weight and fecundity, they remained suitable for use in infection experiments, responding comparably to controls when challenged with Escherichia coli.
By enhancing handling and experimental safety without altering pathogen susceptibility, this method provides a practical refinement for researchers using Galleria mellonella as a host model in infection biology.
Pseudomonas aeruginosa acyl‑CoA dehydrogenases and structure‑guided inversion of their substrate specificity
Meng Wang, Prasanthi Medarametla, Thales Kronenberger, Tomas Deingruber, Paul Brear, Wendy Figueroa, Pok‑Man Ho, Thomas Krueger, James C. Pearce, Antti Poso, James G. Wakefield, David R. Spring & Martin Welch
Nature Communications (Mar 2025) — DOI: 10.1038/s41467‑025‑57532‑z
Why this matters
This collaborative study between researchers at the University of Cambridge and GMRC identifies the key fatty acyl‑CoA dehydrogenases that Pseudomonas aeruginosa uses to oxidise fatty acids and demonstrates how the substrate specificity of these enzymes can be altered through structure‑guided engineering. P. aeruginosa is a major opportunistic human pathogen, and understanding its metabolism helps reveal vulnerabilities that could be exploited in antimicrobial strategies.
The work characterises two dominant enzymes, FadE1 and FadE2, showing that each prefers different fatty acid chain lengths based on structural features of their active sites. By mutating specific residues in the substrate‑binding pockets, the researchers were able to invert the enzymes’ substrate specificities, highlighting fundamental principles of enzyme selectivity.
Importantly, in Galleria mellonella infection experiments, mutants lacking these fatty acyl‑CoA dehydrogenases showed impaired virulence, demonstrating the in vivo relevance of these metabolic pathways and underscoring the utility of Galleria as a host model for pathogenicity studies.
By integrating structural biology, enzymology, genetics, and host infection data, this study provides molecular insight into bacterial fatty acid metabolism and virulence and highlights potential targets for future antimicrobial development.
Meng Wang, Prasanthi Medarametla, Thales Kronenberger, Tomas Deingruber, Paul Brear, Wendy Figueroa, Pok‑Man Ho, Thomas Krueger, James C. Pearce, Antti Poso, James G. Wakefield, David R. Spring & Martin Welch
Nature Communications (Mar 2025) — DOI: 10.1038/s41467‑025‑57532‑z
Why this matters
This collaborative study between researchers at the University of Cambridge and GMRC identifies the key fatty acyl‑CoA dehydrogenases that Pseudomonas aeruginosa uses to oxidise fatty acids and demonstrates how the substrate specificity of these enzymes can be altered through structure‑guided engineering. P. aeruginosa is a major opportunistic human pathogen, and understanding its metabolism helps reveal vulnerabilities that could be exploited in antimicrobial strategies.
The work characterises two dominant enzymes, FadE1 and FadE2, showing that each prefers different fatty acid chain lengths based on structural features of their active sites. By mutating specific residues in the substrate‑binding pockets, the researchers were able to invert the enzymes’ substrate specificities, highlighting fundamental principles of enzyme selectivity.
Importantly, in Galleria mellonella infection experiments, mutants lacking these fatty acyl‑CoA dehydrogenases showed impaired virulence, demonstrating the in vivo relevance of these metabolic pathways and underscoring the utility of Galleria as a host model for pathogenicity studies.
By integrating structural biology, enzymology, genetics, and host infection data, this study provides molecular insight into bacterial fatty acid metabolism and virulence and highlights potential targets for future antimicrobial development.
Characterising phagocytes and measuring phagocytosis from live Galleria mellonella larvae
Jennie S. Campbell, James C. Pearce, Attila Bebes, Arnab Pradhan, Raif Yuecel, Alistair J. P. Brown, James G. Wakefield
Virulence (Dec 2024) — DOI: 10.1080/21505594.2024.2313413
Why this matters
This study develops a novel live‑cell flow cytometry method to characterise haemocytes - the innate immune cells of Galleria mellonella larvae - and measure phagocytosis during infection. Galleria is increasingly used as an in vivo replacement model for mammalian infection studies, but traditional readouts (e.g., melanisation, survival) provide limited insight into immune cell dynamics.
The method reveals that Galleria hemocytes form a single resolvable population based on size and complexity, and that up to 80 % of these cells show phagocytic activity when exposed to fluorescent particles. This provides researchers with a quantitative way to assess cellular immune responses over time in a live host.
Using this assay, the authors demonstrate that Candida albicans cell wall β‑1,3‑glucan masking can subvert hemocyte phagocytosis, underscoring how pathogen traits influence host immune engagement in real time.
By enabling rapid, live analysis of innate immune cell function, this approach broadens the toolkit available for infection biology in Galleria, strengthening its utility as a scalable mammalian replacement model organism.
Jennie S. Campbell, James C. Pearce, Attila Bebes, Arnab Pradhan, Raif Yuecel, Alistair J. P. Brown, James G. Wakefield
Virulence (Dec 2024) — DOI: 10.1080/21505594.2024.2313413
Why this matters
This study develops a novel live‑cell flow cytometry method to characterise haemocytes - the innate immune cells of Galleria mellonella larvae - and measure phagocytosis during infection. Galleria is increasingly used as an in vivo replacement model for mammalian infection studies, but traditional readouts (e.g., melanisation, survival) provide limited insight into immune cell dynamics.
The method reveals that Galleria hemocytes form a single resolvable population based on size and complexity, and that up to 80 % of these cells show phagocytic activity when exposed to fluorescent particles. This provides researchers with a quantitative way to assess cellular immune responses over time in a live host.
Using this assay, the authors demonstrate that Candida albicans cell wall β‑1,3‑glucan masking can subvert hemocyte phagocytosis, underscoring how pathogen traits influence host immune engagement in real time.
By enabling rapid, live analysis of innate immune cell function, this approach broadens the toolkit available for infection biology in Galleria, strengthening its utility as a scalable mammalian replacement model organism.
A microinjection protocol for the greater waxworm moth, Galleria mellonella
Pearce JC, et al. bioRxiv (Sept 2024) — DOI: 10.1101/2024.09.17.613528
Why this matters
This preprint presents a detailed, optimised microinjection protocol for Galleria mellonella embryos, designed to support genetic manipulation and developmental studies in this invertebrate model. Reliable microinjection techniques are essential for introducing DNA, RNA, or genome‑editing reagents into embryos or early stages, but until now, standardised methods for Galleria have been lacking. This work fills that gap by providing a reproducible approach to obtain large numbers of staged embryos and to dechorionate and inject them with precision, improving the efficiency of genetic experiments in the wax moth host.
By enabling consistent access to early developmental stages and facilitating reagent delivery, this protocol supports advances in gene editing, transgenesis, and functional studies in Galleria. Such capabilities are foundational for expanding the model’s utility in infection biology, host–pathogen interaction research, and broader in vivo experimentation that require manipulation of gene function.
The availability of a clear procedural reference helps standardise methods across labs, reducing technical variability and making Galleria mellonella an even more robust and accessible alternative to traditional vertebrate models. GMRC continues to refine and optimise the method of microinjection for transgenesis and CRISPant generation.
Pearce JC, et al. bioRxiv (Sept 2024) — DOI: 10.1101/2024.09.17.613528
Why this matters
This preprint presents a detailed, optimised microinjection protocol for Galleria mellonella embryos, designed to support genetic manipulation and developmental studies in this invertebrate model. Reliable microinjection techniques are essential for introducing DNA, RNA, or genome‑editing reagents into embryos or early stages, but until now, standardised methods for Galleria have been lacking. This work fills that gap by providing a reproducible approach to obtain large numbers of staged embryos and to dechorionate and inject them with precision, improving the efficiency of genetic experiments in the wax moth host.
By enabling consistent access to early developmental stages and facilitating reagent delivery, this protocol supports advances in gene editing, transgenesis, and functional studies in Galleria. Such capabilities are foundational for expanding the model’s utility in infection biology, host–pathogen interaction research, and broader in vivo experimentation that require manipulation of gene function.
The availability of a clear procedural reference helps standardise methods across labs, reducing technical variability and making Galleria mellonella an even more robust and accessible alternative to traditional vertebrate models. GMRC continues to refine and optimise the method of microinjection for transgenesis and CRISPant generation.