Galleria Mellonella Larvae as an In vitro Model for Testing Microbial Pathogenicity

Galleria mellonella is a cosmopolitan insect species that occurs on all continents except Antarctica (Kwadha et al. 2017; Wojda et al. 2020). Because it feeds on beeswax, it is considered a pest. As a holometabolous species, it is characterized by the four phases in its life cycle: egg, larva, pupa and adult or moth stage (Fasasi and Malaka 2006; Kwadha et al. 2017; Pereira et al. 2018; Piatek et al. 2021). The larval form has been used as a model organism in which to study host-pathogen relationships, to test new drugs, and to identify virulence factors of bacterial pathogens (Pereira et al. 2018; Piatek et al. 2021; Chen and Keddie 2021; Ménard et al. 2021).

The G. mellonella larval model is becoming ever more popular, as reflected in the increasing the number of incoming publications containing the term “Galleria mellonella” cited on PubMed (61 publications in 2010 compared with 348 in 2022). The insects used can be divided into two classes. The so-called ‘research’ class is reared without hormones or antibiotics and has a standardized microbiome, whereas the ‘bait’ class is commercially available and is used as bait in fisheries or as pet food (Kwadha et al. 2017; Allonsius et al. 2019; Wojda et al. 2020). The larvae range in size from 1 to 3 cm, which facilitates the injection of compounds and the recovery of organs, tissues and hemolymph for further study (Fasai and Malaka 2006; Kwadha et al. 2017). G. mellonella is characterized by a short life cycle of approximately 5 weeks from the egg to the adult form (Fasai and Malaka 2006; Ramarao and Lereclus 2012; Firacative et al. 2020). Larvae are faster growth, cheaper, and easier to house over model mice. Moreover, G. mellonella larvae do not have nociceptors, so in vitro studies can be undertaken without the approval of a bioethics committee (Ménard et al. 2021). It is noteworthy that the entire genome of the insect has been sequenced, which opens the possibility of its use in research based on genetic modification (Lange et al. 2018). Another major advantage of the organism is the insect's immune system, specifically its humoral response. This, combined with the fact that G. mellonella larvae can be raised at 37°C, allows the temperature conditions in the human body to be simulated, affording an excellent model organism that is capable of simulating infections by human pathogens (Desalermos et al. 2012; Sheehan et al. 2018).

Aims of presented review are to informed potential G. mellonella larvae model users about exemplars of Gram-negative, Gram-positive, fungus pathogenicity and antimicrobials studies. Basic information about G. mellonella immune system are provided.

A scoring system based on several health indices is applicable in studies of human pathogens in G. mellonella larvae (Table I) (Loh et al. 2013).

The nonspecific immune responses of invertebrates protect them from adverse external factors and pathogens. Studies of the genome of G. mellonella larvae have shown that their humoral immunity shares similarities with the human humoral system, which involves pathogen recognition patterns and the body's defense responses. The immune system of G. mellonella consists of the cuticle and the cellular and humoral immune responses. The cuticle is the organism's first line of defense. It is composed of chitin, matrix proteins, and lipids, and its main function is to protect the organism against the entry of pathogens. If the structure of the cuticle is damaged, the cellular and humoral defense factors of the organism are activated (Tao et al. 2021). The cellular immune response is characterized by the involvement of hemocytes, which are phagocytic cells. Hemocytes occur in the insect hemolymph, which has a similar function to vertebrate blood, and are involved in phagocytosis, encapsulation, and nodulation. There are six types of hemocytes in G. mellonella larvae: granulocytes, coagulocytes, plasmocytes, spherulocytes, oenocytoids, and prohemocytes. Granulocytes first attack any microorganism that has entered the body, after which plasmocytes trigger cell layering, resulting in the encapsulation and nodulation of the pathogen. The mechanism of phagocytosis is similar to the cellular immune response of human blood cells (Tsai et al. 2016). The humoral immune response is mainly regulated by soluble effectors, including melanin, opsonins, and antimicrobial peptides (AMPs), which mediate melanization and hemolymph coagulation (Tao et al. 2021).

G. mellonella produces plasma proteins that function in opsoninzation, and recognition, and bind to microbial structures. Apolipophorin III (apoLp-III) is one of such proteins, and plays a key role in the innate immune response of invertebrates. It has high affinity for bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and shares high homology with the mammalian protein E (apoE), responsible for phagocytosis, the detoxification of LPS, and the release of nitric oxide (NO) from thrombocytes. ApoLp-III binds to fungal conidia and β-1,3-glucans to facilitate cell encapsulation. The protein has other important functions in the immune response, including in stimulating hemocytes to secrete superoxide, enhancing the antimicrobial effect of hemolymph, and stimulating the activity of cercopin, another AMP. ApoLp-III influences the permeabilization of the bacterial cell membrane, particularly of Gram-negative bacteria, by interacting with the lysozyme found in G. mellonella larvae (Tsai et al. 2016).

AMPs are important factors in the specific immune response of G. mellonella. They are present at various concentrations in both healthy and infected larvae, primarily in the salivary glands, gastrointestinal and reproductive tracts, fat body, and hemocytes (Ménard et al. 2021). Both cationic and anionic AMPs are present (Trevijano-Contador and Zaragoza 2018). Cationic AMPs can be divided into three groups: peptides with glycine and/or proline residues (gloverin), α-helical linear peptides (moricin, cepropin), and peptides with disulfide bridges (galiomycin, galerimycin) (Ménard et al. 2021). Anionic AMPs are classified as either peptide 1 (AP1) or peptide 2 (AP2). AP2 is present in the hemolymph of both infected and uninfected larvae, and in both cases, the amount of AP2 is constant and relatively high. The efficacy of AP2 against yeast and Micrococcus luteus is low (Sowa-Jasiłek et al. 2020). AMPs induce the efflux of ions from the bacterial cell, which leads to its lysis. Each type of peptide has a different mechanism of action. For example, moricins and cercopins determine the formation of pores in the cytoplasmic membrane, whereas proline- and glycine-rich peptides contribute to the inhibition of the synthesis of key membrane-associated proteins, increasing membrane permeability (Ménard et al. 2021). Undoubtedly, the most important and common feature characterizing AMPs is their ability to destroy microbial cells although AMPs differ in their affinities for filamentous fungi and Gram-positive and Gram-negative bacterial cells (Vertyporokh and Wojda 2017).

Many bacterial pathogens that cause infections in humans are characterized by the secretion of thermolysin-like metalloproteinases (M4 family), which degrade both human and larval defense system proteins. G. mellonella larvae produce inhibitors of thermolysin-like metalloproteinases, the production of which is stimulated by the presence of pathogenic microorganisms. Until now, it is the only known insect inhibitor of microorganismal metalloproteinases, and is involved in the immune response of invertebrates to pathogens (Ménard et al. 2021).

The lysozymes belong to a family of antimicrobial proteins that are very similar to peptidoglycan-degrading muramidases, and act against Gram-positive bacteria and to a lesser extent against Gram-negative bacteria (Ménard et al. 2021). Lysozymes are present in the larval hemolymph, and their concentrations increase after pathogen infection (Sheehan et al. 2021). These proteins interact with opsonins (apoLp-III), in a complex that damages Escherichia coli cells (Zdybicka-Barabas et al. 2013). As well as participating in the immune response, lysozymes also regulate the microbiome of G. mellonella larvae. It has been shown that the microbiomes of larvae in which lysozyme production is limited are dominated by Gram-negative Enterobacteriaceae species (Johnston and Rolff 2015).

Another important mechanism involved in the organism's defense against fungi and bacteria is the melanization pathway (Jorjao et al. 2018; Pereira 2018). During the body's defense response, bacterial components (LTA, LPS) are bound by soluble effector molecules, leading to the release of pro-phenyl oxidase by oenocytoids. The pro-phenyl oxidase is then activated via a serine protease cascade to phenyl oxidase, which oxidizes phenolic compounds. The oxidation reaction generates quinone compounds, which are broken down to melanin, leading to the formation of dark spots on infected larvae. Factors affecting the melanization process include the virulence of the bacterial strain and the number of microorganisms present (inoculum). The melanization phenomenon was observed in G. mellonella larvae infected with an enterotoxigenic E. coli strain, whereas no changes in larval color was observed after infection with a nonpathogenic E. coli strain. The infection of larvae with a suspension of Staphylococcus aureus (10⁶ colony-forming units [CFU]) led to their complete and rapid melanization, although this effect was not observed after infection with 10⁴ CFU (Ménard et al. 2021).

Pseudomonas aeuroginosa strain PA14 is characterized by its strong virulence against G. mellonella larvae. A few bacterial cells were sufficient to kill half of the organisms tested (LD₅₀) (Jander et al. 2000). P. aeuroginosa strains are human pathogens, responsible for various diseases, including ventilator-associated pneumonia (VAP). The genes expressed in P. aeruginosa strains isolated from patients with and without VAP were compared, and no statistically significant differences were detected in the expression of the P. aeruginosa genes associated with biofilm formation or virulence. The only difference noted between the strains was in pigment production. In the VAP-associated strain, pyoverdin was expressed more strongly than pyocyanin, whereas in the non-VAP strain, pyocyanin was expressed more strongly than pyoverdin. Furthermore, the expression of the rhlI and rhlR genes was also weaker in non-VAP strain (Alonso et al. 2020). Pyoverdin production is dependent on the exposure time and the concentration of copper and other metal ions in the bacterial environment (Lear et al. 2022). P. aeuroginosa mutants have been tested in the G. mellonella model. One study tested the effect of the lptE gene on bacterial virulence. About three bacterial cells of wild-type P. aeuroginosa strain PA01 were required as the 90% lethal dose (LD₉₀), whereas the LD₉₀ of the lptE-depleted mutant was 9000–10000 times higher than that of the wild type (Lo Sciuto et al. 2018). In another study the virulence of Legionella pneumophila mutants was tested in the G. mellonella model. A mutant depleted of the flaA gene, which encodes the flagellin protein, did not differ from the L. pneumophila wild-type strain in its replication or virulence in G. mellonella larvae (Harding 2013).

A virulence analysis of 71 E. coli isolates from urinary-tract infections was performed in G. mellonella larvae. The LD₅₀ for each isolate was determined. A low LD₅₀ correlated positively with the expression of the papAH, papC, papEF, sfaS, bmaE, gafD, and kpsMTIII genes (Alghoribi et al. 2014). Expression of the afa/dra, ompT, fimH, fyuA, usp, traT, pap, kpsII, and malx genes correlated with higher mortality (Ciesielczuk et al. 2015). Mutation of the cpx gene, responsible for the stress response, reduced the virulence potential of the bacterium (Leuko and Raivio 2012).

Acinetobacter baumanii strains, which are responsible for infections in healthcare facilities, have also been tested in the G. mellonella model. The most virulent strains are characterized by a enhanced capacity for biofilm formation (Khalil et al. 2021). The expression of A. baumanii virulence factors was shown to be temperature-dependent (Peleg et al. 2009). No differences in insect mortality after infection with several A. baumanii strains were observed in the G. mellonella model, suggesting the rapid adaptability of the bacteria to new environmental conditions (Chapartegui-González and Lázaro-Díez 2018). G. mellonella has been used as a model organism to compare the virulence of two strains of Klebsiella pneumoniae, with or without carbapenemase activity. Observation for 24 h showed that the strains that produced carbapenemases were responsible for the death of 50% of larvae, whereas only 25% of larvae were killed by carbapenemase-negative strains (McLaughlin et al. 2014). Francisella sp. are nonmotile, nonsporulating, gram-negative coccobacilli. causes a zoonotic disease by inhalation of an extremely low infectious dose of bacterial cells (Ahmad et al. 2010). Francisella-infected G. mellonella larvae were used as a model in which to study the efficacy of the antibiotic azithromycin. Galleria mellonella larvae were infected with F. tularensis or F. novicida strains. After incubation for 2 h, the caterpillars were injected with phosphate-buffered saline (PBS), ciprofloxacin, or azithromycin. None of the Francisella-infected insects survived beyond 100 h, whereas survival in the uninfected control group was > 300 h. The mean survival time of larvae administered ciprofloxacin was > 74 h, whereas in the group treated with azithromycin, the mean survival time increased to > 160 h. The study clearly demonstrated the efficacy of azithromycin in the treatment of Francisella infection (Ahmad et al. 2010).

Enterococci have been recognized as being among the most common hospital-acquired pathogens. G. mellonella was used to assess the pathogenic potential of the Enterococcus faecalis proteins extracellular gelatinase (GelE) and serine protease (SprE). To analyze the bacterial virulence mechanisms, purified GelE and SprE enzymes were injected directly into the insect hemolymph. GelE showed lytic activity against the AMP cecropin of G. mellonella, a defense factor that acts in the early stages of microbial infection. In contrast to GelE, the SprE protease showed no activity against the insect immune system (Park et al. 2007).

Staphylococcus aureus the Gram-positive opportunistic pathogen, is one of the most common causatives of nosocomial infections. Study virulence of S. aureus were done in a dose-effect manner. The infection of G. mellonella larvae with 1 × 10⁷ CFU cells of S. aureus in suspension resulted in 100% mortality after 24 h. When the dose was reduced by two orders of magnitude (1 × 10⁵), mortality was reduced to 80%. The survival of test insects was also shown to decrease with increasing temperature (Peleg et al. 2009). S. aureus can also exist as small-colony variant (SCVs), which are essential in establishing antibiotic resistance. SCVs have an intracellular survival pattern that allows them to evade the host immune system and antibacterial substances (Zheng et al. 2021). However, they grow more slowly and have lower virulence than prototype S. aureus, as demonstrated in studies in the G. mellonella model. In one study, tests were performed on three S. aureus strains (JP310, JP1450, and JP1486) and their SCV counterparts (Zheng et al. 2021). The survival of G. mellonella larvae after inoculation for 120 hours with the SCV of strain JP310 was reduced to 50% of that of larvae injected with the native prototype; with strains JP1450 and JP1486, larval survival reduction, after injection with the corresponding SCVs, was about 40% and 50%, respectively, after observation for 5 days (Zheng et al. 2021).

Listeria monocytogenes is an invasive foodborne Gram-positive pathogen. G. mellonella has also proved a useful model for the virulence analysis of L. monocytogenes. A suspension of the bacterium (1 × 10⁷ cells) showed 100% reproducible lethality against G. mellonella larvae. The virulence of L. monocytogenes was also temperature-dependent, and increasing the temperature from 30°C to 37°C increased in the average survival rate of G. mellonella larvae. A kinetic analysis indicated that in the first 2 h of the experiment, the number of bacteria decreased, but increased rapidly thereafter. Above results were observed when bacteria were injected to hemolymph not for oral application. An expression analysis of the genes responsible for virulence in L. monocytogenes showed that they were expressed at similar levels in G. mellonella larvae as in mammalian organisms after infection (Joyce and Gahan 2010).

G. mellonella larvae have been used to analyze the virulence of the fungus Metarhizium robertsii, a microorganism capable of producing proteases that are resistant to inhibitors present in the host. These enzymes include chymotrypsin-like and subtilisin-like proteases and metalloproteases. The immune response of larvae to infection with M. robertsii is regulated by epigenetic processes, including the expression of microRNAs, which are responsible for controlling posttranscriptional protein synthesis, histone deacetylation by histone deacetylases, and histone acetylation by histone acetyltransferases. M. robertsii produces toxic thermolysin, a member of the M4 family of metalloproteinases, which includes a number of virulence factors responsible for infections. Several genes of Metarhizium are responsible for thermolysin bisynthesis. Small amounts of fungal thermolysin are sufficient to activate the immune system of G. mellonella larvae, and this thermolysin induces the formation of larval peptides that induce inflammation (Mukherjee and Vilcinskas 2018).

Candida albicans strains are present in the microbiomes of the gastrointestinal tract. It is an opportunistic pathogen. The cell wall of C. albicans is composed of chitin, mannoproteins, phospholipomannan, and β-1,6- and β-1,3-glucans. The chitin and glucans form an internal rigid protective layer, which gives shape to the cell. Mannoproteins, in contrast, are present in the outer layer of the cell wall and are involved in the adhesion of the fungus to host tissues, leading to the activation of the host's immune system. During invasion by C. albicans, both the cellular and humoral immune response mechanisms are activated in the host. The key proteins and peptides involved in this process include defensins, LL-37, histatins, and lysozyme. G. mellonella larvae are increasingly used as a model organism in which to study the pathogenicity of C. albicans and the efficacy of antifungal preparations. Several studies have shown that the exposure of the larvae to the fungus triggers the activation of the humoral immune response, including the production of defense proteins and peptides, which are also involved in the formation of immune memory (Sowa-Jasiłek et al. 2016).

In an era of increasing antibiotic resistance, new antibacterial compounds are urgently required. The activities of 90 extracts from native Atlantic Forest tree species were tested against S. aureus in G. mellonella larvae. The results suggested that prenylated flavonoids and isoflavones are effective anti-staphylococcal agents (Chagas Almeida et al. 2019). In another study, compound HEScL from the leaves of Syzygium cumini, combined with silver nanoparticles, exerted both time- and dose-dependent bactericidal and antibiofilm effects (Kaul et al. 2022).

Both natural and synthetic compounds have been studied in the G. mellonella larval model. The low-molecular-weight peptide NapFFKK-OH forms a hydrogel, the toxicity properties of which were tested in vivo in the G. mellonella model. No in vivo toxicity or death was observed in G. mellonella larvae within the 5 days of the experiment at NapFFKK-OH concentrations of ≤ 2% w/v. However, treatment with 2% (w/v) NapFFKK-OH reduced S. aureus by 4.4 log10 CFU/mL after 72 h (McCloskey et al. 2019). Another study showed that chromone 5-maleimide substitution compound CM3a is toxic to both S. aureus and G. mellonella larvae. CM3a effectively eradicated an S. aureus biofilm by reducing bacterial cell viability and exerting low-level toxicity. Therefore, chromium derivatives of CM3a may offer an alternative treatment for infections caused by S. aureus (Qing et al. 2021). New antimicrobial molecules are being sought with various approaches, including the dual-host method, which is used to identify and validate new anti-methicillin-resistant S. aureus (MRSA) compounds. In one study, five new compounds, PPT, NNC, TBB, GW4064, and PD198306, positively affected the survival of the test organisms. The dual-host method can be used to identify compounds with both antimicrobial activity and relatively low toxicity against the eukaryotic cells of G. mellonella larvae, which are potentially very valuable therapeutic agents (Khader et al. 2020).

The utility of a staphylococcal phage (monophage Sb-1) and a mixture of bacteriophages (PYO) has also been demonstrated in the G. mellonella model. The authors suggested that bacteriophages are effective against infections of MRSA strains. PYO was more effective than Sb-1 against S. aureus, with a higher survival rate among the infected insects. The timing of bacteriophage injection also influenced the efficacy of the therapy. Bacteriophages were injected 1 h before or after infection. There was less mortality in the infected larvae in which the bacteriophages were injected before infection (Tkhiaishvili et al. 2020). G. mellonella larvae were also used to assess the activity of bacteriophage 191219 against an S. aureus biofilm. This phage effectively destroyed the bacterial cells in vitro, depending on the dose used (Mannala et al. 2022). The study also confirmed the utility of a preparation of Staphylococcus monophage Sb-1, which is highly efficacious against S. aureus, including antibiotic-resistant isolates. In another study, the efficacy of this preparation in controlling or preventing S. aureus colonization of medical foreign bodies (K-wires) was tested in G. mellonella larvae. The bacteria were reduced in preparations treated with the Sb-1/daptomycin combination and the Sb-1 preparation prevented the colonization of K-wires by S. aureus, as did vancomycin (Materazzi et al. 2022).

With the discovery of potential new therapeutic agents, novel therapies are also being sought. One such therapy is photodynamic therapy (PDT), which shows antimicrobial activity against certain pathogens. In one study, G. mellonella larvae were used as the model organism in which to evaluate the effectiveness of PDT and to investigate the regulation of humoral immunity by PDT. 5-Aminolevulinic acid (ALA) was used as the photosensitizer. The study showed that ALA-PDT exerted a defensive effect against bacterial infection by inducing the humoral immune responses in larvae (Huang et al. 2020).

The development of novel strategies that increase the efficacy of antibiotics by exerting synergistic effects can lead to the preventing of antibiotic resistance in bacteria. For example, the synergistic effects of the antibiotics cefazolin and fosfomycin against S. aureus were investigated in the G. mellonella model. The addition of as little as 0.8 mg/kg fosfomycin to cefazolin restored cefazolin sensitivity of MRSA strains (Kussmann et al. 2021).

Selected examples of studies of antibacterial and antifungal compounds tested in the G. mellonella larvae model are shown in Tables II and III.

The use of G. mellonella larvae as a model in which to analyze the pathogenicity of microorganisms and to evaluate the efficacy of new drugs has great potential utility and benefits. The literature review presented here shows that G. mellonella offers a practical, low-cost, and ethically acceptable research tool that can be used in various fields of medicine and biology. In addition, correlation between bacterial virulence in insects and mammals models can be tested, as was presented with P. aeruginosa mutant (Jander et al. 2020).

In studies of the pathogenicity of Gram-negative bacteria, such as E. coli, A. baumanii, and K. pneumoniae, the use of the G. mellonella model has allowed the analysis of pathogen-host interactions and the impact of virulence factors on larval survival. Similarly, studies of Gram-positive bacteria, such as S. aureus and E. faecalis, and the fungi C. albicans and M. robertsii based on this model have provided important information on their pathogenicity.

G. mellonella caterpillars are also used as an in vitro model in which to evaluate the efficacy of new drugs and improve existing therapies. This experimental system makes it possible to test the antipathogenic activities of various chemical compounds and natural substances.

From this literature review, it can be concluded that G. mellonella is a versatile model for testing microbial pathogenicity and evaluating new drugs. The ease of breeding, rapid developmental cycle, complete immune system, and fully sequenced genome of G. mellonella make it a valuable research tool. Unfortunately, the model still suffers from a lack of standardization. Insects from nonstandardized commercial cultures, which do not have standardized microbiomes, can negatively affect the reproducibility of research. As was pointed out standardized rearing – temperature, humidity, diet, light period are crucial for high quality G. mellonella larvae for microbiological studies (De Jong et al. 2022).