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Larval morphology. (a) Healthy Galleria mellonella larvae. (b) Larvae killed as a result of Aspergillus fumigatus infection 48 h previously. Note the dark colour of cadavers due to melanisation.
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Insects are convenient models for assessing the virulence of microbial pathogens or for assessing the -efficacy of antimicrobial drugs and give results comparable to those that can be obtained using mammals. Galleria mellonella larvae are easy to purchase and inoculate and provide results within 48 h. Various parameters may be used to monitor the e...
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... Assess larvae at regular intervals (every 2 h) for viability and disease progression. For assessment of viability, larvae should be gently probed with a needle, and if no response is observed, the larvae may be considered to be dead. Changes in cuticle melanisation can also be used to monitor the severity of an infection ( Fig. 2) (see Note 4). 1. Pierce the backs of the anterior end ("head") of three ran- domly chosen larvae with a sterile needle and collect the yellow haemolymph ("blood") into a single pre-chilled tube contain- ing a few grains of 1-phenyl-3-(2-thiazolyl)-2-thiourea (see Notes 5 and ...
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Citations
... The greater wax moth, Galleria mellonella, is a member of the order Lepidoptera and is mostly known as a pest of beehives [1]. As G. mellonella can grow for several generations on artificial food [2], are easily inoculated with bacteria, can grow at 37 °C, and have few ethical issues, it has gained in popularity as a model host, in particular to study microbial interactions such as pathogenesis [3,4]. Previously, most studies have used larvae supplied from pet food shops, which may have been grown with antibiotics and hormones, but recently there have been from BioSystems Technology (TruLarv™), and also from a local Norfolk beekeeper. ...
Objective
Study of the human infant gut microbiome requires the use of surrogate mammalian species such as mice. We sought to investigate the usefulness of the greater wax moth larva, Galleria mellonella, as an alternative.
Results
We have analysed the native gut microbiome of Galleria and developed methods for clearing the native microbiome and introducing species from human infant faecal samples. We find that some species, e.g. enterococci, are more successful at recolonisation, but that others, e.g. Bifidobacterium, are less so. The work paves the way for using Galleria rather than mice in this and similar work.
... Sixth instar larvae of G. mellonella (SAGIP, Italy), weighing 0.3-0.4 g, were selected for experimental use (Fallon et al. 2012). 10 6 spores in a volume of 20 µl were injected per larva through one of the hind pro-legs as described previously (Kelly and Kavanagh 2011). ...
Mucorales are basal fungi that opportunistically cause a potentially fatal infection known as mucormycosis (black fungus disease), which poses a significant threat to human health due to its high mortality rate and its recent association with SARS-CoV-2 infections. On the other hand, histone methylation is a regulatory mechanism with pleiotropic effects, including the virulence of several pathogenic fungi. However, the role of epigenetic changes at the histone level never has been studied in Mucorales . Here, we dissected the functional role of Set1, a histone methyltransferase that catalyzes the methylation of H3K4, which is associated with the activation of gene transcription and virulence. A comparative analysis of the Mucor lusitanicus genome (previously known as Mucor circinelloides f. lusitanicus ) identified only one homolog of Set1 from Candida albicans and Saccharomyces cerevisiae that contains the typical SET domain. Knockout strains in the gene set1 lacked H3K4 monomethylation, dimethylation, and trimethylation enzymatic activities. These strains also showed a significant reduction in vegetative growth and sporulation. Additionally, set1 null strains were more sensitive to SDS, EMS, and UV light, indicating severe impairment in the repair process of the cell wall and DNA lesions and a correlation between Set1 and these processes. During pathogen-host interactions, strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo. Our findings suggest that the histone methyltransferase Set1 coordinates several cell processes related to the pathogenesis of M. lusitanicus and may be an important target for future therapeutic strategies against mucormycosis.
... We used the G. mellonella infection model to test the antifungal efficacy of HAL in vivo, as G. mellonella utilizes phagocytic cells (hemocytes) as part of their host defense [34,35]. We divided the G. mellonella larvae into four groups, with ten larvae in each group: (1) a control group receiving no drug treatment, (2) a group treated with 0.5 mg/kg HAL, (3) a group treated with 1 mg/kg HAL, and (4) a group treated with 2 mg/kg HAL. ...
Candida albicans, a prominent opportunistic pathogenic fungus in the human population, possesses the capacity to induce life-threatening invasive candidiasis in individuals with compromised immune systems despite the existence of antifungal medications. When faced with macrophages or neutrophils, C. albicans demonstrates its capability to endure oxidative stress through the utilization of antioxidant enzymes. Therefore, the enhancement of oxidative stress in innate immune cells against C. albicans presents a promising therapeutic approach for the treatment of invasive candidiasis. In this study, we conducted a comprehensive analysis of a library of drugs approved by the Food and Drug Administration (FDA). We discovered that halofantrine hydrochloride (HAL) can augment the antifungal properties of oxidative damage agents (plumbagin, menadione, and H2O2) by suppressing the response of C. albicans to reactive oxygen species (ROS). Furthermore, our investigation revealed that the inhibitory mechanism of HAL on the oxidative response is dependent on Cap1. In addition, the antifungal activity of HAL has been observed in the Galleria mellonella infection model. These findings provide evidence that targeting the oxidative stress response of C. albicans and augmenting the fungicidal capacity of oxidative damage agents hold promise as effective antifungal strategies.
... One such potential model is a model of larvae of Galleria mellonella, the greater wax moth [20]. The larva model has become popular recently for its benefits in studying virulence factors of bacterial and fungal pathogens [20][21][22]. Its similarity in terms of innate immunity to that of vertebrates and its ability to be maintained at the physiological body temperature (37-40 • C) make G. mellonella a suitable surrogate model for microbial infection studies [20,23]. ...
Coccidioidomycosis (CM) can manifest as respiratory and disseminated diseases that are caused by dimorphic fungal pathogens, such as Coccidioides species. The inhaled arthroconidia generated during the saprobic growth phase convert into multinucleated spherules in the lungs to complete the parasitic lifecycle. Research on coccidioidal virulence and pathogenesis primarily employs murine models typically associated with low lethal doses (LD100 < 100 spores). However, the Galleria model has recently garnered attention due to its immune system bearing both structural and functional similarities to the innate system of mammals. Our findings indicate that Coccidioides posadasii can convert and complete the parasitic cycle within the hemocoel of the Galleria larva. In Galleria, the LD100 is between 0.5 and 1.0 × 106 viable spores for the clinical isolate Coccidioides posadasii C735. Furthermore, we demonstrated the suitability of this model for in vivo antifungal susceptibility tests to validate the bioreactivity of newly discovered antifungals against Coccidioides. Additionally, we utilized this larva model to screen a Coccidioides posadasii mutant library showing attenuated virulence. Similarly, the identified attenuated coccidioidal mutants displayed a loss of virulence in a commonly used murine model of coccidioidomycosis. In this study, we demonstrated that Galleria larvae can be applied as a model for studying Coccidioides infection.
... Unlike Drosophila melanogaster, wild-type larvae of the greater wax moth Galleria mellonella are susceptible to infection with multiple pathogenic fungi [87][88][89], making them convenient model hosts of invasive fungal diseases and obviating the need for genetic manipulation. Galleria larvae are inexpensive to purchase and maintain, and the infection model is technically undemanding [88]. ...
... Unlike Drosophila melanogaster, wild-type larvae of the greater wax moth Galleria mellonella are susceptible to infection with multiple pathogenic fungi [87][88][89], making them convenient model hosts of invasive fungal diseases and obviating the need for genetic manipulation. Galleria larvae are inexpensive to purchase and maintain, and the infection model is technically undemanding [88]. Sixth instar Galleria mellonella larvae weighing 0.3-0.4 ...
... Sixth instar Galleria mellonella larvae weighing 0.3-0.4 g are used [88]. For experiments requiring drug treatment, dosage is determined by measuring the hemolymph volume of larvae of various weights and calculating the mean hemolymph volume/kg weight by linear regression [90]. ...
Mucormycosis presents a formidable challenge to clinicians and researchers. Animal models are an essential part of the effort to decipher the pathogenesis of mucormycosis and to develop novel pharmacotherapeutics against it. Diverse model systems have been established, using a range of animal hosts, immune and metabolic perturbations, and infection routes. An understanding of the characteristics, strengths, and drawbacks of these models is needed to optimize their use for specific research aims.
... The greater wax moth, Galleria mellonella, is a member of the order Lepidoptera and is mostly known as a pest of beehives [1]. As G. mellonella can grow for several generations on arti cial food [2], are easily inoculated with bacteria, can grow at 37°C, and have few ethical issues, it has gained in popularity as a model host, in particular to study microbial interactions such as pathogenesis [3,4]. Previously, most studies have used larvae supplied from pet food shops, which may have been grown with antibiotics and hormones, but recently there have been attempts to standardise G. mellonella as a model [5,6], including TruLarv™ [7]. ...
Objective
Study of the human infant gut microbiome requires the use of surrogate mammalian species such as mice. We sought to investigate the usefulness of the greater wax moth larva, Galleria mellonella, as an alternative.
Results We have analysed the native gut microbiome of Galleria and developed methods for clearing the native microbiome and introducing species from human infant faecal samples. We find that some species, e.g. enterococci, are more successful at recolonisation, but that others, e.g. Bifidobacterium, are less so. The work paves the way for using Galleria rather than mice in this and similar work.
... 6. To ground the larvae, introduce them to each of the corresponding labeled treatment tubes. Then, using the pestle for microcentrifuge tube [15], homogenize the larval tissue for 5 min and mix it using a vortex mixer for 1 min. ...
Galleria mellonella larva has been widely exploited as an infection model for bacteria and fungi. Our laboratory uses this insect as a model for fungal infection caused by the genus Malassezia, in particular, systemic infections caused by Malassezia furfur and Malassezia pachydermatis, which are poorly understood. Here, we describe the G. mellonella larva inoculation process with M. furfur and M. pachydermatis and the posterior assessment of the establishment and dissemination of the infection in the larvae. This assessment was done through the evaluation of larval survival, melanization, fungal burden, hemocytes populations, and histological changes. This methodology allows for the identification of virulence patterns between Malassezia species and the impact of inoculum concentration and temperature.Key wordsAlternative animal modelGalleria mellonella larva
Malassezia furfur
Malassezia pachydermatis
Systemic infection modelingFungemia
... 16 Alternatively to higher mammalians, invertebrates such as Drosophila melanogaster, Caenorhabditis elegans, zebrafish, and G. mellonella have been widely used as infection models to study host-pathogen interactions as well as virulence of bacterial and fungal pathogens. [17][18][19][20] In addition, those models enabled testing of antimicrobial agents and drugs. These models are economical, ethically legitimate and easy to handle. ...
Fungal implant-associated bone infections are rare but difficult to treat and often associated with a poor outcome for patients. Candida species account for approximately 90% of all fungal infections. In vivo biofilm models play a major role to study biofilm development and potential new treatment options, however, there are only a very few in vivo models to study fungi-associated biofilms. Furthermore, mammalian infection models are replaced more and more due to ethical restrictions with other alternative models in basic research. Recently, we developed an insect infection model with Galleria mellonella larvae to study biofilm-associated infections with bacteria. Here, we further expanded the G. mellonella model to study in vivo fungal infections using Candida albicans and Candida krusei. We established a planktonic and biofilm-implant model to test different anti-fungal medication with amphotericin B, fluconazole and voriconazole against the two species and assessed the fungal biofilm-load on the implant surface. Planktonic infection with C. albicans and C. krusei showed the killing of the G. mellonella larvae at 5x105 CFU. Treatment of larvae with anti-fungal compounds with amphotericin B and fluconazole showed significant survival improvement against planktonic C. albicans infection, but voriconazole had no effect. Titanium and stainless steel K-wires were pre-incubated with C. albicans and implanted inside the larvae to induce the biofilm infection on the implant surface. The survival analysis revealed significantly reduced survival of the larvae with Candida spp. infection compared to non-infected implants. The treatment with antifungal amphotericin B and fluconazole resulted in a slight and non-significant improvement survival of the larvae. The treatment with the anti-fungal compounds in the biofilm-infection model was not as effective as in the planktonic infection model, which highlights the resistance of fungal biofilms to anti-fungal compounds like in bacterial biofilms. Scanning electron microscopy (SEM) analysis revealed the formation of a fungal biofilm with hyphae and spores associated with larvae tissue on the implant surface. Thus, our study highlights the use of G. mellonella larvae as alternative in vivo model to study biofilm-associated implant fungal infections and that fungal biofilms exhibit high resistance profiles comparable to bacterial biofilms. The model can be used in the future to test anti-fungal treatment options for fungal biofilm infections. This article is protected by copyright. All rights reserved.
... A. baumannii WT or hdc::TOPO mutant strains, in their exponential phase, were collected, washed in PBS, and then diluted to the indicated cell density, as determined by the optical density at 600 nm. Ten microliter of the inoculum was injected into the hemocoel of each larva through the last left proleg [18]. Bacterial colony counts on LB agar plates were used to confirm all inocula. ...
... Hemocytes were pelleted by centrifugation (500 g, 5 min) at room temperature. The cells were washed once with IPS and finally resuspended in PBS containing 5 mM glucose [18]. ...
We found that histamine (10<sup>−9</sup> M) did not have any effect on the in vitro capture of Escherichia coli by neutrophils but accelerated its intracellular killing. In contrast, histamine (10<sup>−6</sup> M) delayed the capture of Escherichia coli by neutrophils and reduced the amounts of pHrodo zymosan particles inside acidic mature phagosomes. Histamine acted through the H<sub>4</sub>R and the H<sub>2</sub>R, which are coupled to the Src family tyrosine kinases or the cAMP/protein kinase A pathway, respectively. The protein kinase A inhibitor H-89 abrogated the delay in bacterial capture induced by histamine (10<sup>−6</sup> M) and the Src family tyrosine kinase inhibitor PP2 blocked histamine (10<sup>−9</sup> M) induced acceleration of bacterial intracellular killing and tyrosine phosphorylation of proteins. To investigate the role of histamine in pathogenicity, we designed an Acinetobacter baumannii strain deficient in histamine production (hdc::TOPO). Galleria mellonella larvae inoculated with the wild-type Acinetobacter baumannii ATCC 17978 strain (1.1 × 10<sup>5</sup> CFU) died rapidly (100% death within 40 h) but not when inoculated with the Acinetobacter baumannii hdc::TOPO mutant (10% mortality). The concentration of histamine rose in the larval haemolymph upon inoculation of the wild type but not the Acinetobacter baumannii hdc::TOPO mutant, such concentration of histamine blocks the ability of hemocytes from Galleria mellonella to capture Candida albicans in vitro . Thus, bacteria-producing histamine, by maintaining high levels of histamine, may impair neutrophil phagocytosis by hijacking the H<sub>2</sub>R.
... However, due to ethical standards, time, costs, and legal restrictions, several insect species have been explored for use as a novel in vivo models or pre-model organisms for physiological, toxicological, microbiological, and clinical studies instead of mammalian species (Lionakis and Kontoyiannis 2005, Rowan et al. 2009, Kavanagh and Fallon 2010, Desbois and Coote 2011, Junqueira 2012, MacCallum et al. 2013, Browne et al. 2014, Jaconsen 2014, Maurer et al. 2015, Wojda 2017, Cutuli et al. 2019. One such example of a model insect species is the greater wax moth, Galleria mellonella Linnaeus (Lepidoptera; Pyralidae) which is preferred due to its anatomical and physiological characteristics, high reproductive ability, and ease of culturing under laboratory conditions (Fallon et al. 2012, Browne and Kavanagh 2013, Cook and Mcarthur 2013. Additionally, G. mellonella's toxicity reports and innate immune defense mechanisms have significant similarities with vertebrate innate immune responses. ...
Immunotoxic effects of sodium benzoate (SB, E211), sodium nitrate (SNa, E251), and sodium nitrite (SNi, E250), a few of the most common food preservatives, on the model organism Galleria mellonella L. (Lepidoptera: Pyralidae) larvae were investigated in this study. The last instar larvae were used for all experimental analyses. For this purpose, median lethal doses of SB, SNa, and SNi were applied to the larvae by the force-feeding method. We found that force-feeding G. mellonella larvae with SB, SNa, and SNi significantly reduced the larval total hemocyte counts, prohemocyte, and granulocyte ratios but increased plasmatocyte, spherulocyte, and oenocyte ratios, as well as the hemocyte mitotic indices and micronucleus frequency. The spreading ability of hemocytes and hemocyte-mediated immune responses were lower in the SB, SNa-, and SNi-treated larval groups compared to controls. Apoptotic indices were higher in all larval groups treated with food preservatives, but increments in necrotic indices were only significantly higher in SNi-treated larvae compared to controls. Our research shows that SB, SNa, and SNi have immunotoxic and cytotoxic potential on G. mellonella larvae. Thus, we suggest that G. mellonella larvae can be used as preliminary in vivo models to screen the immunotoxic effects of food preservative agents.
