Biology subjects

Wakefield, J. G.

Publications and source records attributed to Wakefield, J. G..

5 recordsLinked to original sources

bioRxiv2026

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

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 {degrees}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 -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. HighlightsO_LICold-shock at -20 {degrees}C for 10 minutes inhibits silk extrusion. C_LIO_LILarvae survive treatment with no loss of suitability for infection studies. C_LIO_LIDevelopment slows and adult weight, fecundity, and wing quality decline. C_LIO_LISilk glands stay intact; gene expression remains unchanged after cold-shock. Cytoskeletal disruption likely drives the failure of silk secretion. C_LI

microbiology↗

bioRxiv2024

A microinjection protocol for the greater waxworm moth, Galleria mellonella

A limitation to the non-vertebrate 3Rs model Galleria mellonella has been the lack of genetic toolkit. A common requirement for genetic tractability is a method to introduce exogenous material to the unicellular embryo, the most common of which is microinjection. This short article describes a detailed method for rearing Galleria mellonella to collect large amounts of staged embryos and to dechorionate and microinject embryos with limited mortality. Research HighlightsO_ST_ABSScientific BenefitsC_ST_ABSMicroinjection allows the introduction of a wide variety of substances, such as DNA, RNA or drugs into Galleria embryos, providing the technology needed for genetic engineering, gene editing and functional studies in this important model organism. 3Rs BenefitsGalleria is being increasingly used as a partial animal replacement model, especially in the field of infection biology. However, uptake has been limited by the lack of genetic and molecular tools. This protocol takes a step towards removing these barriers by providing a means to introduce substances that can create transgenic or genetically engineered Galleria. Practical BenefitsProtocol for injecting substances into Galleria, using for the most part easily accessible equipment. Current ApplicationsGenerating stable transgenic and gene-edited Galleria lines. Potential ApplicationsAny technique requiring the introduction of substances to Galleria embryos. This includes applying existing techniques such as pBac-mediated transgenesis or CRISPR/Cas-based gene-editing to this organism,in order to generate engineered strains of Galleria. It could also include injection of synthetic mRNAs encoding proteins fused to fluorescent genes (such as GFP) in order to visualise their dynamics in living embryos; and the injection of drugs that perturb particular cell or developmental processes in order to learn more about early Galleria development.

bioengineering↗

bioRxiv2024

PiggyBac mediated transgenesis and CRISPR/Cas9 knockout in the greater waxmoth, Galleria mellonella

The larvae of the greater waxmoth, Galleria mellonella, are gaining prominence as a versatile non-mammalian in vivo model to study host-pathogen interactions. Their ability to be maintained at 37{degrees}C, coupled with a broad susceptibility to human pathogens and a distinct melanisation response that serves as a visual indicator for larval health, positions Galleria as a powerful resource for infection research. Despite these advantages, the lack of genetic tools, such as those available for zebrafish and fruit flies, has hindered development of the full potential of Galleria as a model organism. In this study, we describe a robust methodology for generating transgenic Galleria using the PiggyBac transposon system and for precise gene knockouts via CRISPR/Cas9 technology. These advances significantly enhance the utility of Galleria in molecular research, opening the way to its widespread use as an inexpensive and ethically compatible animal model for infection biology and beyond.

bioengineering↗

bioRxiv2023

Characterising phagocytes and measuring phagocytosis from live Galleria mellonella larvae

Over the last 20 years, the larva of the greater waxmoth, Galleria mellonella, has rapidly increased in popularity as an in vivo mammalian replacement model organism for the study of human pathogens. Despite this, experimental readouts of response to infection are generally limited to observing the melanisation cascade - where the organism turns black as part of the systemic immune response - and quantifying larval death over time. As an invertebrate, Galleria harbour an innate immune system comprised of both humoral components and a repertoire of innate immune cells - termed hemocytes. Though information on subtypes of hemocytes exist, there are conflicting reports on their exact number and function. Flow cytometry has previously been used to assay Galleria hemocytes, but protocols include both centrifugation and fixation - physical methods which have the potential to affect hemocyte morphology prior to analysis. Here, we present a method for live hemocyte analysis by flow cytometry, revealing that Galleria hemocytes constitute only a single resolvable population, based on relative size or internal complexity. Using fluorescent zymosan particles, we extend our method to show that up to 80% of the Galleria hemocyte population display phagocytic capability. Finally, we demonstrate that the developed assay reliably replicates in vitro data, showing that cell wall {beta}-1,3-glucan masking by Candida albicans subverts phagocytic responses. As such, our method provides a new tool with which to rapidly assess phagocytosis and understand live infection dynamics in Galleria.

cell biology↗

bioRxiv2020

Intimate functional interactions between TGS1 and the Smn complex revealed by an analysis of the Drosophila eye development

Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). In addition, TGS1 depletion results in increased hTR levels and telomere elongation in human cells. Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to devise new therapies for SMA and diseases caused by telomerase insufficiency.

genetics↗