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      Standardization of G. mellonella Larvae to Provide Reliable and Reproducible Results in the Study of Fungal Pathogens

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          Abstract

          In the past decade, Galleria mellonella (wax moth) larvae have become widely used as a non-mammalian infection model. However, the full potential of this infection model has yet to be realised, limited by the variable quality of larvae used and the lack of standardised procedures. Here, we review larvae suitable for research, protocols for dosing larvae, and methods for scoring illness in larvae infected with fungal pathogens. The development of standardised protocols for carrying out our experimental work will allow high throughput screens to be developed, changing the way in which we evaluate panels of mutants and strains. It will also enable the in vivo screening of potential antimicrobials at an earlier stage in the research and development cycle.

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          Galleria mellonella infection models for the study of bacterial diseases and for antimicrobial drug testing

          abstract Galleria mellonella (greater wax moth or honeycomb moth) has been introduced as an alternative model to study microbial infections. G. mellonella larvae can be easily and inexpensively obtained in large numbers and are simple to use as they don't require special lab equipment. There are no ethical constraints and their short life cycle makes them ideal for large-scale studies. Although insects lack an adaptive immune response, their innate immune response shows remarkable similarities with the immune response in vertebrates. This review gives a current update of what is known about the immune system of G. mellonella and provides an extensive overview of how G. mellonella is used to study the virulence of Gram-positive and Gram-negative bacteria. In addition, the use of G. mellonella to evaluate the efficacy of antimicrobial agents and experimental phage therapy are also discussed. The review concludes with a critical assessment of the current limitatons of G. mellonella infection models.
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            Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms.

            Our knowledge of gene function has increasingly lagged behind gene discovery, hindering our understanding of the genetic basis of microbial phenotypes. Recently, however, massively parallel sequencing has been combined with traditional transposon mutagenesis in techniques referred to as transposon sequencing (Tn-seq), high-throughput insertion tracking by deep sequencing (HITS), insertion sequencing (INSeq) and transposon-directed insertion site sequencing (TraDIS), making it possible to identify putative gene functions in a high-throughput manner. Here, we describe the similarities and differences of these related techniques and discuss their application to the probing of gene function and higher-order genome organization.
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              Galleria mellonella as a model system to study Cryptococcus neoformans pathogenesis.

              Evaluation of Cryptococcus neoformans virulence in a number of nonmammalian hosts suggests that C. neoformans is a nonspecific pathogen. We used the killing of Galleria mellonella (the greater wax moth) caterpillar by C. neoformans to develop an invertebrate host model system that can be used to study cryptococcal virulence, host immune responses to infection, and the effects of antifungal compounds. All varieties of C. neoformans killed G. mellonella. After injection into the insect hemocoel, C. neoformans proliferated and, despite successful phagocytosis by host hemocytes, killed caterpillars both at 37 degrees C and 30 degrees C. The rate and extent of killing depended on the cryptococcal strain and the number of fungal cells injected. The sequenced C. neoformans clinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformans genes previously shown to be involved in mammalian virulence (CAP59, GPA1, RAS1, and PKA1) also played a role in G. mellonella killing. Combination antifungal therapy (amphotericin B plus flucytosine) administered before or after inoculation was more effective than monotherapy in prolonging survival and in decreasing the tissue burden of cryptococci in the hemocoel. The G. mellonella-C. neoformans pathogenicity model may be a substitute for mammalian models of infection with C. neoformans and may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies.
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                Author and article information

                Journal
                J Fungi (Basel)
                J Fungi (Basel)
                jof
                Journal of Fungi
                MDPI
                2309-608X
                06 September 2018
                September 2018
                : 4
                : 3
                : 108
                Affiliations
                [1 ]Biosystems Technology Ltd., 1 Colleton Crescent, Exeter EX2 4DG, UK; o.champion@ 123456biosystemstechnology.com
                [2 ]College of Life and Environmental Science, University of Exeter, Stocker Road, Exeter EX4 4QD, UK; s.bates@ 123456exeter.ac.uk
                Author notes
                [* ]Correspondence: r.w.titball@ 123456exeter.ac.uk ; Tel.: +44-1392-725157
                Author information
                https://orcid.org/0000-0002-0162-2077
                https://orcid.org/0000-0003-0489-5705
                Article
                jof-04-00108
                10.3390/jof4030108
                6162639
                30200639
                3f0ee71d-4c67-414f-b60b-4b16ba94e8d2
                © 2018 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 13 August 2018
                : 05 September 2018
                Categories
                Review

                galleria mellonella,infection model,fungi,genome,pathological score,end point

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