Galleria mellonella Immune System: Comparison
Please note this is a comparison between Version 1 by Letizia Angiolella and Version 2 by Peter Tang.

The study of pathogenicity and virulence of fungal strains, in vivo in the preclinical phase, is carried out through the use of animal models belonging to various classes of mammals (rodents, leproids, etc.). Although animals are functionally more similar to humans, these studies have some limitations in terms of ethics (animal suffering), user-friendliness, cost-effectiveness, timing (physiological response time) and logistics (need for adequately equipped laboratories). A good in vivo model must possess some optimal characteristics to be used, such as rapid growth, small size and short life cycle. For this reason, insects, such as Galleria mellonella (Lepidoptera), Drosophila melanogaster (Diptera) and Bombyx mori (Lepidoptera), have been widely used as alternative non-mammalian models. Due to their simplicity of use and low cost, the larvae of G. mellonella represent an optimal model above all to evaluate the virulence of fungal pathogens and the use of antifungal treatments (either single or in combination with biologically active compounds). A further advantage is also represented by their simple neuronal system limiting the suffering of the animal itself, their ability to survive at near-body ambient temperatures as well as the expression of proteins able to recognise combined pathogens following the three R principles (replacement, refinement and reduction). 

  • Galleria mellonella
  • fungi
  • Aspergillus spp.
  • Candida spp.
  • virulence factors

1. Introduction

The experimental use of animals is extremely important in science, especially for the development of new antimicrobial drugs with enhanced safety and efficacy [1]. However, at such a preclinical phase, in vivo mammalian models, primarily mice and rats, have some disadvantages, such as the need for adequate infrastructure and lengthy experiments. In addition, they always pose a number of critical issues due to the identification and use of animal models complying with the ethical, experimental and legislative principles recommended by the European directive on animal protection guided by the three R rules (i.e., replacement, reduction and refinement) [2][3][2,3]. In recent years, however, many in vivo studies have used insects, which approximately account for 90% of all animal species. The insect immune system shares many features with human innate defence; therefore, it can be called “evolutionary roots of human innate immunity” [4]. For this reason, insects are used not only in studies of the interactions with their natural pathogens but also in studies of the virulence factors of human pathogens as well as in tests of antimicrobial drugs in vivo [5][6][7][5,6,7]. Therefore, insects, such as Galleria mellonella (Lepidoptera), Drosophila melanogaster (Diptera) and Bombyx mori (Lepidoptera), have been widely used as alternative non-mammalian models. Table 1 shows the differences among the invertebrate models utilised in fungal infections.
Table 1.
In vivo infection models with invertebrates.
Thanks to its rapid life cycle, cost-effectiveness and advanced technology availability, Drosophila melanogaster, commonly known as the fruit fly, is a model organism used to study a wide range of disciplines, from fundamental genetics to tissue and organ development [8]. The D. melanogaster genome is 60% homologous to that of humans and since about 75% of the genes responsible for human diseases have homologues in flies [9], these insects have recently become useful tools for studying human diseases, including rare Mendelian diseases [10], neurodegenerative diseases [11] and cancer [12]. The molecular mechanisms of pathogenic proteins encoded by viral and bacterial genomes have also been studied in Drosophila [13].
B. mori, also known as the silkworm, is often used as an infection model. This is due to the availability of germ plasm banks, which maintain genetic stock collections; these centres adopt an artificial diet, thus contributing to standardising the quality of the supply of this insect [14]. The organs and systems of B. mori and mammals are anatomically similar, thus making this insect a valuable model organism for studying various life science processes. This has been made possible by the availability of the complete sequence of its genome and the development of technologies for genetic manipulation. Finally, B. mori is still widely used in sericulture and biotechnology as a bioreactor for producing recombinant proteins and silk-based biomaterials [15].
Alternative in vivo models, such as Galleria mellonella, have been studied [16][17][18][19][16,17,18,19]; moreover, this insect has been widely used in experiments to evaluate the toxic potential and antimicrobial activity of drugs, including antifungal agents [20].
G. mellonella is also a suitable model for studying the expression of virulence factors and host–pathogen interactions, such as the innate immune response to microorganisms, thus representing the first step in human health studies [21]. One of its most important characteristics is its innate immune system, whose functional structures are similar to those of mammals [22].
The treatment of infections caused by fungal pathogens is extremely challenging both due to the presence of MDR (multidrug-resistant) strains mainly infecting immunocompromised patients and the limited availability of antifungal drugs, which are highly toxic [23]. The most studied fungi are Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans, which cause a high mortality rate [24][25][26][27][24,25,26,27].

2. Galleria mellonella Model

G. mellonella, a species belonging to the order Lepidoptera and part of the Pyralidae family, is a ubiquitous parasite in the hives of bees, wasps and bumblebees that feeds on honey, beeswax, bee faeces, pollen and cocoons. With a holometabolous life cycle, this insect has five stages of development and therefore a very short life cycle: egg, caterpillar, pre-pupa, pupa and adult. Figure 1 reports the life cycle of G. mellonella in different stages and the overall timeframe. The eggs develop into caterpillars after 5–8 days, and in about 6 weeks, the caterpillar matures and grows. The larval stage has a cylindrical elongated form measuring 16–20 mm. After 8 to 10 moults taking place from day 28 to 6 weeks, the caterpillar stops feeding and maintains slight motility, but in the meantime, the development of a silk cocoon begins—this is the pre-pupa stage [28][29][28,29]. The pre-pupa subsequently matures into a pupa, immobilised in the cocoon [30]. The adult form appears after a period ranging from 4 to 8 weeks; the adult moth has a reddish-brown colour and is active in the nocturnal phase. In its adult form, G. mellonella can lay up to 300 eggs, although some studies report a higher number of up to 600 [31][32][33][34][35][36][31,32,33,34,35,36]. The life cycle from the hatching of the eggs to the maturation of the larvae may be affected by nourishment and temperature (the optimal one being between 28 and 30 °C) [37]. Its quick and easy life cycle requires no special attention. Unlike other insects, the larvae of G. mellonella survive at temperature ranges that vary from 15 °C to 37 °C. At 37 °C, human physiological conditions can be mimicked, and this is necessary to reach a temperature at which the virulence factors of pathogens can be expressed [5][38][5,38].
Figure 1.
Life cycle of
Galleria mellonella
: (1) eggs; (2) caterpillar; (3) pre−pupa; (4) pupa; (5) adult form.
The nervous system of G. mellonella, like all Lepidoptera, is very simple from a functional and anatomical point of view, thus enabling theour group of scientists to reconstruct it using computer software (Amira 5.3.3) [39]. In the larval form, we have the following subdivisions of the body: head, three thoracic segments with two legs and an abdomen consisting of ten segments. The abdomen has eight prolegs and two anal prolegs [40]. The larval body has an internal cavity with an open circulatory system with a digestive system, which originates with a simple masticatory mouthpart (with attached salivary glands) and ends with an anal opening. Furthermore, ventrally, reswearchers find a nervous system characterised by ganglia and various neuronal connections [41]. This system makes the organism sensitive to abiotic factors such as light, temperature and humidity [39]. Sexual polymorphisms can be identified in the adult form. The male emits ultrasound as a form of mating call and is smaller and beige in colour; meanwhile, the female is larger, with a wider wingspan and releases pheromones (non-anal), useful for attracting the male [38]. Experimental research aimed at sequencing the genome of G. mellonella has been recently conducted, and from the analyses carried out using PacBio technologies and genomic libraries, it was realised that the DNA of larvae has a modest amount of genetic homologies with the genomes of humans, rats and other organisms. These features promote and encourage the use of Lepidoptera as a model in biomedical research [22].

3. G. mellonella Immune System

The immune system of G. mellonella is organised in an innate form that can be divided into humoral and cellular responses. The latter, unlike the former, presents a series of homologies with the immune systems of mammals [22], a characteristic confirmed by a series of experimental tests [42][43][44][45][42,43,44,45]. This innate immune system is the first line of defence used by the insect against various pathogens, as also seen in vertebrates [42]. The ability of the insect to exploit the cuticle is mainly due to its structure. The endocuticle, the innermost layer, contains chitin fibrils, while the outermost layer, the epicuticle, contains fatty acids, lipids and sterols [43]. This creates a dense and resistant barrier against pathogens and harmful mechanical forces, but it can be damaged or degraded [44]. Another system that participates in nonspecific immune action is the haemolymph, equivalent to mammalian blood; therefore, it is responsible for the transport of various substances (e.g., nutrients, signal molecules and waste) [45].

3.1. Cellular Immune System

G. mellonella haemocytes, such as plasmatocytes and granulocytes, due to their adherent properties, are phagocytic cells. Haemocytes are responsible for the phagocytosis, encapsulation and nodulation of the invading pathogen. Haemocytes may either be free in the haemolymph or associated with specific internal organs (especially the digestive, reproductive and cardiac ones) [46][47][46,47]. The haemocytes are able to recognise pathogens through specific receptors present on their surface: the PRRs (pathogen recognition receptors). The pathogenic antigens that are recognised by these receptor structures are PAMPs, which include lipopolysaccharides (LPS), peptidoglycans, lipoteichoic acids (LTA) and β-1,3 glucan [48]. The recognition molecules in question include apolipophorin–III (ApoLp III), which is capable of identifying fungal β-1,3 glucan as well as bacterial LPS and LPA [48]. Haemolin does not have direct antibacterial properties, although it binds to the lipoteichoic acid of Gram-positive bacteria and to the lipopolysaccharides (LPS) of Gram-negative bacteria [49]. Table 2 shows the different haemocytes and their functions [46][50][46,50].
Table 2.
Different types of haemocytes and their functions.
. The melanisation process is activated by surface receptors, which recognise specific molecular patterns. Among the receptors able to recognise β-1,3-glucan, LPS and peptidoglycans, there is also C-reactive protein, a homologue of TLRs in mammals [51]. This is released and carried either to the cuticle, the damaged site or the encapsulated pathogen until the polymerisation of melanin is generated [51].

3.2. Humoral Immune System

The humoral immune response of G. mellonella involves various processes and molecular responses, which do not include antibodies (as in mammals) but rather simple antimicrobial peptides (AMPs). The immediate contact with microorganisms (bacteria, fungi, viruses, protozoa) determines the transcription of genes for the synthesis of AMPs [52]. AMPs are polypeptide chains of 10–40 residues playing a fundamental role in host defence and which are produced mainly in body fat in both the digestive and reproductive tract to be subsequently released into the haemolymph. They are produced in high concentrations in the first six hours of the infection and then decrease after 3 days. They can be divided into anionic or cationic forms and based on their structure, and they can be either linear α-helices, peptides with a structure stabilised by disulfide bridges or peptides with glycine and/or proline residues [53][54][55][56][53,54,55,56]. Table 3 reports all the peptides involved in humoral response.
Table 3.
Components of humoral response in
Galleria mellonella
Encapsulation comprises the development of capsules enveloping and blocking a possible no-self agent, which is internalised in the insect. Encapsulation occurs when pathogens are too large for phagocytosis. The response of the host to microbial invasion is characterised by the development of nodules, called nodulation [38]. The process starts with granular cells attacking the surface of the microbes. This triggers the release of multiple plasmatocyte-spreading peptides, attacking the surface of bacteria, fungal spores or foreign targets, resulting in the formation of a smooth capsule [47]. Melanisation does not occur in this process. Melanisation, as the main defence mechanism against a high range of microorganisms and comprising the deposition of melanin in the haemolymph of the pathogen, will be followed by the coagulation of the haemolymph and opsonisation in order to kill the pathogen [50]. This process begins on the cuticle surface of the larvae with simple black spots, gradually spreading to the entire cuticular surface if the infection becomes more severe. This melanisation may involve the entire larva, to the point of making it completely black; and the latter condition is synonymous with a serious infection, causing the death of the larvae [39]
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