Immune response of Galleria mellonella after injection with non-lethal and lethal dosages of Candida albicans
Graphical abstract
Introduction
Elucidation of various aspects of insect defence mechanisms requires the use of both natural and opportunistic pathogens in infection experiments. The former allow understanding the immune response as a specific defence strategy directed against a natural pathogen, which has appeared as a result of host-pathogen co-evolution (Keebaugh and Schlenke, 2014). At the same time, studies of insect immune response to infection with opportunistic pathogens should not be ignored, as they can provide information about the general and universal properties of defence mechanisms. Insects possess only innate immunity, which shares many common features with the innate defence in vertebrates (Buchmann, 2014). These arthropods attract growing attention as convenient models for testing human pathogen virulence and drug efficiency in vivo (Cutuli et al., 2019). One of the best insect models to study the properties of innate defence is the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae). Its ca. 6-week life cycle (at the optimal temperature) and the fact that one female can lay approx. 2000 eggs allow for efficient breeding. Also, the relatively large size of Galleria larvae facilitate injection and collection of hemolymph and other organs for further analysis (Wojda, 2017). G. mellonella live in beehives or, more often, in stored waxes, where the larvae feed on honey, pollen, and wax and cause galleriasis (Gulati and Kaushik, 2004, Kwadha et al., 2017, Williams, 1997). Larvae undergo seven moulting stages before pupation and are the stage most often used for injection experiments (Kwadha et al., 2017). Imagos live only ca. 12 d (females) or 21 d (males) and do not feed; their only role is reproduction. Galleria, like all insects, is protected by a chitin-containing integument. The internal organs of ectodermic origin such as the trachea, foregut, and hindgut, are covered by cuticle, protecting the insects from pathogens. If these barriers are broken, cellular and humoral immune responses can be triggered. Galleria plasmatocytes and granulocytes are able to engulf intruding bacteria, while larger foreign bodies and groups of microorganisms are entrapped in capsules and nodules; thus, isolating pathogens from the rest of the insect's body (Cytryńska et al., 2016). Molecular patterns of pathogens are recognised by receptors activating signalling pathways, mainly Toll and Imd. Imd functions exclusively in immunity, whereas the Toll pathway also regulates insect development (Viljakainen, 2015). The Imd pathway is triggered by most Gram-negative bacteria, and the Toll pathway is activated by Gram-positive bacteria, fungi, and danger signals (Ming et al., 2014). Gram-positive bacteria with lysine-type peptidoglycan are bound by short forms of Peptidoglycan Recognition Proteins: PGRP-SA and PGRP-SD, which leads to the activation of the Toll pathway. In turn, Gram-negative and some Gram-positive bacteria, e.g. Bacillus, containing DAP-type peptidoglycan (composed of diaminopimelic acid), are recognized by the so-called long forms of PGRPs: PGRP-LC and PGRP-LE (Charroux et al., 2009), which in turn activate the Imd pathway. Signalling through both pathways leads to the activation of transcription factors Dif and Relish, respectively, which are homologues of the human NF-κB factor (Lu et al., 2019a, Sheehan et al., 2018, Wang et al., 2019). As a result, effector molecules of humoral immunity, e.g. antimicrobial peptides (AMPs), appear in the hemolymph to kill the invading microorganisms mostly by destruction of their membranes (Wu et al., 2018). The main source of defence molecules is the fat body, an organ with very high metabolism and an analogue of the mammalian liver (Arrese and Soulages, 2010). In addition, the JAK/STAT pathway, which regulates many biological processes involving immunity, participates in hematopoiesis and cellular immunity, regulation of defence against viral infections, gut immunity, general stress response and wound healing (Bang, 2019, Wu et al., 2018). Synthesis of the dark pigment melanin is regulated by phenol oxidase (PO); the non-active form, prophenol oxidase (PPO), is released from oenocytoids after infection or injury. Melanin can be deposited either on the surface of invading microorganisms to facilitate recognition/killing of the pathogen or on the clot, which is thus hardened until the injured epidermis is restored (Eleftherianos and Revenis, 2011, Hillyer, 2016). The transcriptomes of infected and non-infected G. mellonella were compared by Vogel et al. (2011), revealing immune-regulated genes. Recently, the entire genome of the greater wax moth was published, which will contribute to identification of other molecules involved in the immunity of this animal (Lange et al., 2018).
One of the opportunistic organisms that can cause the death of G. mellonella larvae is the yeast-like fungus Candida albicans (Bergin et al., 2006, Marcos-Zambrano et al., 2019, Vertyporokh et al., 2019). C. albicans is often present as part of the natural microbiome of various organisms, including humans. In immune-compromised patients, it causes a disease called candidiasis. C. albicans has the ability to switch from the yeast-type to the filamentous type of growth, depending on ambient conditions. This feature contributes to the virulence of the fungus (Singkum et al., 2019). Many other virulence factors enabling C. albicans to colonise both vertebrate and invertebrate organisms have been identified. These include adhesins, invasins, and extracellular hydrolytic enzymes useful for invasion of host tissues (Schaller et al., 2005, Staniszewska et al., 2012, Wibawa, 2016). Although C. albicans is not a natural insect pathogen, a non-lethal dosage introduced into the hemocel of G. mellonella can induce enhanced resistance to repeated infection with the same fungus (Vertyporokh et al., 2019). This means that the insect immune system is able to “remember” a previous infection and protects the organism more efficiently when it encounters the pathogen for the second time. What is more, this so-called immune priming phenomenon was found to be specific; pre-infection did not prevent further infection with other organisms (Vertyporokh et al., 2019). Here, we show differences in immune response of G. mellonella to C. albicans depending on the dosage of fungal cells injected into the larval hemocel.
Section snippets
Insects, fungi, and injection
G. mellonella were reared on a natural diet, honeybee nest debris, at 28˚C in darkness. Final instar larvae, 3 d after the last moult and weighing about 200 mg, were used for testing. Candida albicans (strain ATCC 10231) was cultivated on YPD medium (1% yeast extract, 2% bactopepton, 2% glucose) at 30 °C with 120 rpm shaking. The fungi were centrifuged at 5500g and suspended in phosphate buffered saline (PBS, 140 mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4, 1.76 mM KH2PO4 in pH 7.4). The density of
Symptoms of disease
The sensitivity of G. mellonella larvae to the intrahaemocelic injection of C. albicans cells strictly depended on the dose of the fungus. Only 5% of the larvae did not survive the dosage of 2 × 104 cells (LD5, non-lethal dosage). The ten-fold higher dosage caused the death of approx. 50% of the injected animals after 72 hpi, and, ultimately, approx. 95% at 120 h post injection (LD95; lethal dosage). The dosage of 2 × 106 caused very rapid death of all animals (LD100), mostly within the first
Discussion
C. albicans is an opportunistic pathogen that can kill G. mellonella when injected into the larval hemocel. Therefore, the greater wax moth is used as a non-vertebrate model to study pathogenicity, effectiveness of virulence factors, the ability to induce infection by different C. albicans mutants, and in in vivo tests of antifungal drugs (Brennan et al., 2002, Gu et al., 2018, Lu et al., 2019b, Marcos-Zambrano et al., 2019, Pereira et al., 2018, Trevijano-Contador and Zaragoza, 2018).
References (55)
- et al.
Pre-exposure to yeast protects larvae of Galleria mellonella from a subsequent lethal infection by Candida albicans and is mediated by the increased expression of antimicrobial peptides
Microb. Infect.
(2006) - et al.
Hemolymph coagulation and phenoloxidase in Drosophila larvae
Dev. Comp. Immunol.
(2005) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding
Analyt. Biochem.
(1976)- et al.
Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae
FEMS Immunol. Med. Microbiol.
(2002) - et al.
Bacterial detection by Drosophila peptidoglycan recognition proteins
Microb. Infect.
(2009) - et al.
Detection of antibacterial polypeptide activity in situ after sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Anal. Biochem.
(2001) - et al.
In vivo activity of fluconazole/tetracycline combinations in Galleria mellonella with resistant Candida albicans infection
J. Glob. Antimicrob. Resist.
(2018) Insect immunology and hematopoiesis
Dev. Comp. Immunol.
(2016)- et al.
Coevolution of parasitic fungi and insect hosts
Zoology
(2016) - et al.
Insights from natural host-parasite interactions: the Drosophila model
Dev. Comp. Immunol.
(2014)
Purification and cDNA cloning of a cecropin-like peptide from the great wax moth, Galleria mellonella
Mol. Cell.
A different repertoire of Galleria mellonella antimicrobial peptides in larvae challenged with bacteria and fungi
Dev. Comp. Immunol.
Persephone/Spätzle pathogen sensors mediate the activation of Toll receptor signaling in response to endogenous danger signals in apoptosis-deficient Drosophila
J Biol Chem.
Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa
Analyt. Biochem.
Humoral immune response of Galleria mellonella after repeated infection with Bacillus thuringiensis
J. Invert. Pathol.
Host-pathogen interactions upon the first and subsequent infection of Galleria mellonella with Candida albicans
J. Insect Physiol.
Peptidoglycan recognition proteins in insect immunity
Mol. Immunol.
Heat shock affects host-pathogen interaction in Galleria mellonella infected with Bacillus thuringiensis
J. Insect Physiol.
Insect fat body: energy, metabolism, and regulation
Annu. Rev. Entomol.
JAK STAT signaling in insect immunity
Entomol. Res.
Activation of insect phenoloxidase after injury: endogenous versus foreign elicitors
J. Innate Immun.
The potential of the Galleria mellonella innate immune system is maximized by the co-presentation of diverse antimicrobial peptides
Biol. Chem.
Evolution of innate immunity: clues from invertebrates via fish to mammals
Front. Immunol.
Galleria mellonella as a consolidated in vivo model hosts: new developments in antibacterial strategies and novel drug testing
Virulence
Up-Regulation of antimicrobial peptides gallerimycin and galiomicin in Galleria mellonella infected with Candida yeasts Displaying different virulence traits
Mycopathologia
Virulence of Candida albicans mutants toward larval Galleria mellonella (Insecta, Lepidoptera, Galleridae)
Can. J. Microbiol.
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