Cold-Shock-Mediated Inhibition of Silk Extrusion in Galleria mellonella (Lepidoptera: Pyralidae) for Improved Handling in Infection Studies: Effects on Developmental Traits and Pathogen Susceptibility

Abstract The greater wax moth, Galleria mellonella , is an increasingly important invertebrate model for infection biology, yet silk extrusion during handling complicates larval injections and hampers survival assessment. Here, we develop and characterise a simple cold-shock method that reliably inhibits silk production without compromising larval viability. Larvae exposed to −20 °C for 10 minutes completely suppress silk extrusion with 100% survival, representing a substantial improvement over previous chilling approaches. Cold-shocked larvae successfully remained capable of completing development, although pupation and adult emergence were delayed, body weight and fecundity were reduced, and wing deformities were more common. While cold-shock did not alter silk gland morphology, spinneret structure, or fibroin gene expression, confocal imaging revealed pronounced disorganisation of F-actin and α-tubulin networks within silk gland cells, indicating cytoskeletal disruption as a likely mechanism underlying silk inhibition. When challenged with Escherichia coli , cold-shocked larvae responded comparably to controls, with survival influenced primarily by feeding status. Together, these findings demonstrate that short-term cold-shock provides an efficient, reproducible, and easy implemented method for preventing silk extrusion in Galleria larvae, markedly improving handling and experimental safety while preserving their suitability as a model host for pathogen research. Highlights Cold-shock at −20 °C for 10 minutes inhibits silk extrusion. Larvae survive treatment with no loss of suitability for infection studies. Development slows and adult weight, fecundity, and wing quality decline. Silk glands stay intact; gene expression remains unchanged after cold-shock. Cytoskeletal disruption likely drives the failure of silk secretion.