Pathogens and Disease, 78, 2020, ftaa056

doi: 10.1093/femspd/ftaa056
Advance Access Publication Date: 0 2020
Minireview

MINIREVIEW

Galleria mellonella as an infection model: an in-depth

look at why it works and practical considerations for
successful application
Monalessa Fábia Pereira1, *,† , Ciro César Rossi2,† , Giarlã Cunha da Silva3 ,
Jéssica Nogueira Rosa3 and Denise Mara Soares Bazzolli3, *,‡
1
Laboratório de Bioquı́mica e Microbiologia, Departamento de Ciências Biológicas, Universidade do Estado de
Minas Gerais, 36800-000, Carangola, MG, Brazil, 2 Laboratório de Microbiologia Molecular, Departamento de
Microbiologia Médica, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro,
21941-901, Rio de Janeiro, RJ, Brazil and 3 Laboratório de Genética Molecular de Bactérias, Instituto de
Biotecnologia Aplicada à Agropecuária—BIOAGRO, Departamento de Microbiologia, Universidade Federal de
Viçosa, 36570-900, Viçosa, MG, Brazil
∗
Corresponding authors: Denise Mara Soares Bazzolli, Tel: +55-31-3612-2454; E-mail: dbazzolli@ufv.br, and Monalessa Fábia Pereira; E-mail:
monalessa.pereira@uemg.br

One sentence summary: In this review, we describe, with examples, the advantages that make Galleria mellonella a versatile infection model to study
host–bacterial pathogen interactions, and discuss practices aimed at successful experiments.
†
These authors contributed equally to the work.
Editor: Juliana Campos Junqueira
‡
Denise Mara Soares Bazzolli, http://orcid.org/0000-0002-0371-6966

ABSTRACT
The larva of the greater wax moth Galleria mellonella is an increasingly popular model for assessing the virulence of bacterial
pathogens and the effectiveness of antimicrobial agents. In this review, we discuss details of the components of the G.
mellonella larval immune system that underpin its use as an alternative infection model, and provide an updated overview
of the state of the art of research with G. mellonella infection models to study bacterial virulence, and in the evaluation of
antimicrobial efficacy. Emphasis is given to virulence studies with relevant human and veterinary pathogens, especially
Escherichia coli and bacteria of the ESKAPE group. In addition, we make practical recommendations for larval rearing and
testing, and overcoming potential limitations of the use of the model, which facilitate intra- and interlaboratory
reproducibility.

Keywords: Galleria mellonella; animal alternative model; microbial pathogenicity; antimicrobial therapy; 3Rs

INTRODUCTION
The larva of the greater wax moth Galleria mellonella is well
accepted by the scientific community worldwide (Fig. 1) as an
experimental model to study host–pathogen interactions and
the effectiveness of antimicrobial agents (Tsai, Loh and Proft
2016; Cutuli et al. 2019). Over 2000 scientific articles have been
published in PubMed on G. mellonella so far, of which 271 were
published in 2019 alone, demonstrating the growing popular-
ity of this infection model. In particular, there has been an
expansion in the diversity of bacterial pathogens studied, and G.
mellonella–pathogen infection models have made an invaluable

Received: 25 June 2020; Accepted: 18 September 2020


C FEMS 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

1
 2 Pathogens and Disease, 2020, Vol. 00, No. 00
Figure 1. Locations and representativity (%) of institutions that conduct studies with G. mellonella. To build this worldwide overview of G. mellonella research, we
considered the country of the institution to which the corresponding author belongs. The analysis was made after obtaining data from a bibliographic search carried
out in July 2020 at PubMed (MEDLINE database) with articles containing the keyword ‘Galleria mellonella’.
contribution to research with human and veterinary bacterial
pathogens (Tsai, Loh and Proft 2016; Cools et al. 2019).
The use of the G. mellonella model reinforces the impor-
tance of applying the ‘3Rs’ principles (replacement, reduction
and refinement) in animal experimentation, aiming to substi-
tute vertebrate infection models and, consequently, reduce the
number of mammals used in research. As an invertebrate, G. mel-
lonella is not included in animal welfare legislation and ethics
guidelines (Graham and Prescott 2015; Hubrecht and Carter
2019).
The advantages of using the G. mellonella model are numer-
ous (Fig. 2). Rearing G. mellonella is less expensive and easier to
maintain than mammalian models, and does not require major
adaptations to the laboratory’s infrastructure. The large size of
the larvae (12–20 mm) ensures easy handling, and facilitates the
collection of tissues and analysis. The large size of the larvae
also allows the precise quantification of the inocula, which can
be injected directly into the larval hemocoel (Ramarao, Nielsen-
Leroux and Lereclus 2012; Cook and McArthur 2013). Galleria mel-
lonella’s short life cycle makes it suitable for high-throughput
studies (Tsai, Loh and Proft 2016). From our decade-long expe-
rience with this insect, a good-quality diet and careful breeding
can lead to a life cycle of 40–60 days.
Some physiological and immunological characteristics of G.
mellonella are fundamental for the success of the model. Lar-
vae can be kept at 37◦ C, equivalent to the body temperature of
mammalian hosts. As temperature has been shown to affect the
expression of microbial virulence factors, this feature is impor-
tant when assessing virulence of mammal pathogens (Tsai, Loh
and Proft 2016; Twittenhoff et al. 2020). Despite not having an
adaptive immune response, the immune system of G. mellonella
shares many similarities with the mammalian innate immune
response and, as a consequence, can be exploited to assess the
virulence of microbial pathogens and produce results compara-
ble to those obtained with mammalian systems (Sheehan et al.
2018).
Although results depend highly on the target specificity of
the microorganisms’ arsenal of virulence, G. mellonella’s rel-
atively advanced system of antimicrobial defenses makes it
a suitable and informative infection model (López Hernández
et al. 2015). Studies have shown that some virulent bacterial
strains are lethal for G. mellonella as they are for mammalian
hosts, especially mice (Seed and Dennis 2008; Pereira et al. 2015;
Propst et al. 2016). Moreover, for some important pathogenic
species, histopathological effects and cellular responses found
in G. mellonella resemble those found in human infections.
For example, Staphylococcus aureus infection results in nod-
ule formation in G. mellonella with similar structures to those
found in human abscesses (Sheehan, Dixon and Kavanagh
2019). Likewise, histopathological effects caused by Mycobac-
terium abscessus include granulomatous structures as seen in
humans infected with this bacterium (Meir et al. 2018). In addi-
tion to similar pathological effects being found in G. mellonella to
those in conventional mammalian infection models, increased
levels of survival in response to antibacterial treatment have
also been observed in G. mellonella and mice (Jeon et al. 2019;
Leshkasheli et al. 2019).
Despite the numerous advantages offered by G. mellonella for
the study of Gram-positive and Gram-negative bacteria, it is
important to be aware of experimental variables that can influ-
ence the outcome of the study. In addition, the limitations of the
model must be recognized, so that the results can be properly
interpreted. This review will address the advantages and appli-
cations of the model to study bacterial pathogens, and discuss
issues that we consider to be of great relevance for strengthen-
ing the use of G. mellonella as an alternative infection model.
 Pereira et al. 3

Figure 2. Advantages offered by the G. mellonella alternative infection model to study bacterial pathogens. Several aspects of the G. mellonella larva support its application
in in vivo experiments: a high number of repetitions will increase the statistical support of the study; the fact that the larva is relatively large (12–20 mm) allows easy
handling and extraction of tissues and fluids, as well as inoculation of precisely quantified substances and microorganisms directly in the hemocoel; the larvae can be
reared in the laboratory in small containers, provided with natural air flow, without the need for major adaptations in the laboratory structure; since the larval innate
immune system shares similarities with that of mammals, experiments with the insect can, to some extent, replace experiments with vertebrates; a good-quality diet
and careful breeding can lead to a shorter life cycle of 40–60 days; and the larvae can be kept at 37◦ C, equal to the body temperature of mammals and suitable to test
several bacterial pathogens. These advantages allow faster screening of varied compounds with antimicrobial activity before testing in vertebrates.

THE IMMUNE SYSTEM OF G. MELLONELLA
AND THE SUCCESS OF THE MODEL
A primary reason for the success of the G. mellonella model
in the study of microbial virulence is that its immune system
shares a high degree of structural and functional similarity with
that of the innate immune system of vertebrates. The immune
response of G. mellonella consists of two tightly interconnected
components: cellular (cell-mediated) and humoral responses
(Browne, Heelan and Kavanagh 2013).
The cellular response is mediated by hemocytes, cells analo-
gous to human phagocytes, involved with phagocytosis, encap-
sulation and nodulation of the invading agent (Fig. 3). To date, six
types of hemocytes have been identified in G. mellonella: prohe-
mocytes, plasmatocytes, granular cells, coagulocytes, spherulo-
cytes and oenocytoids. Plasmatocytes and granular cells are the
most abundant hemocytes in the hemolymph (Wu et al. 2016).
Hemocytes are found free in the hemolymph or attached to
internal organs, such as the digestive tract, fat body and heart
surface of the insect. The concentration of hemocytes in the
hemolymph varies during the life of the insect and in response
to pathogens (Browne, Heelan and Kavanagh 2013; Pereira et al.
2015; Arteaga Blanco et al. 2017).
The humoral response involves soluble effector molecules
such as antimicrobial peptides (AMPs), complement-like pro-
teins (opsonins), melanin and products of proteolytic cascades,
which immobilize or kill pathogens (Eleftherianos and Revenis
2011; Sheehan et al. 2018).
Proteomic studies have shed light on the great diversity and
dynamics of AMP production (Sheehan, Dixon and Kavanagh
2019; Sheehan et al. 2020), which has been recently shown to be
regulated, in part, by non-coding microRNAs (miRNAs), as it hap-
pens in vertebrates (Mukherjee et al. 2020). AMPs are produced
mainly in the fat body, hemocytes, salivary glands and repro-
ductive tract of G. mellonella in response to microbial invasion.
They play a major role in the insect’s innate immunity, given
their broad-spectrum microbicidal activity. So far, 18 known or
putative AMPs have been identified, some of which are similar
to molecules characterized in mammals, namely: two types of
cecropins, a heliocin-like peptide, an inducible serine protease
inhibitor 2, galiomycin, gallerimycin, gloverin, Gm anionic pep-
tides 1 and 2, Gm proline-rich peptides 1 and 2, lysozyme, five
types of moricin-like peptides and X-tox (Brown et al. 2009; Tsai,
Loh and Proft 2016; Sheehan et al. 2020).
Galleria mellonella produces several opsonins that recognize
and bind to conserved microbial components such as the
lipopolysaccharides (LPS), lipoteichoic acid (LTA) and peptidogly-
can, similar to the pattern recognition receptors (PRRa) found in
mammals.
The opsonin Apolipophorin-III (ApoLp-III) has a high affin-
ity for hydrophobic ligands, such as LPS and LTA. ApoLp-III has
a multifunctional role in the immune system of G. mellonella,
being able to stimulate the production of superoxide by hemo-
cytes, increase the production of cecropin and act synergis-
tically with lysozyme, thus increasing its antimicrobial activ-
ity (Maravilla et al. 2020). ApoLp-III is also involved in recog-
nizing/differentiating the type of pathogen (e.g. Gram-positive
and Gram-negative bacteria, yeasts and filamentous fungi), and
assembling an adequate immune response (Stączek et al. 2018).
The peptidoglycan recognition protein (PGRP), another
opsonin, recognizes peptidoglycan (Wang et al. 2019). In some
insects, PGRPs have been shown to hydrolyze peptidoglycan, but
this has not yet been demonstrated in G. mellonella (Tsai, Loh and
Proft 2016).
GmCP8 is an opsonin produced in the fat body, the midgut
and the integument of G. mellonella. It is secreted in the
hemolymph and binds strongly to LPS, LTA and β-1,3-glucan
(Kim et al. 2010; Barbaro et al. 2019).
The opsonin hemolin, a member of the immunoglobulin
superfamily, binds LPS and LTA, and associates with hemocytes
 4 Pathogens and Disease, 2020, Vol. 00, No. 00

Figure 3. Major components of G. mellonella innate immune system. Galleria mellonella immune system comprises components of a cellular response and a humoral
response. The cellular response comprehends six types of cells (top left): the prohemocytes, plasmatocytes, granulocytes, spherulocytes, oenocytoids and coagulo-
cytes. Some of these cells (but not only those depicted in the top right of the figure) are involved with clearance of invading agents by phagocytosis, nodulation and
encapsulation. Some cells capable of recognizing molecular patterns associated to pathogens (such as the peptidoglycan and lipopolysaccharides, bottom right) trigger
the phenoloxidase pathway, to produce melanin. Melanin is one of the components of the humoral response of the innate immune system of G. mellonella, which also
includes antimicrobial peptides and opsonins (bottom left).

(Aathmanathan et al. 2018). Hemolin can be expressed in the silk
gland and fat body of G. mellonella, and is upregulated during
bacterial and fungal infections (Shaik and Sehnal 2009; Shee-
han et al. 2020). Hemolin transcripts are induced by bacteria or
purified bacterial components, such as the LPS and phorbol 12-
myristate 13-acetate (Aathmanathan et al. 2018).
The melanization process is essential for the defense of G.
mellonella against microbial pathogens, and results in the syn-
thesis and deposition of melanin around the microbe in the
hemolymph (Kavanagh and Reeves 2004). Melanin production is
catalyzed by the enzyme phenoloxidase (PO), which is produced
as the inactive zymogen pro-phenoloxidase (pro-PO) in hemo-
cytes. Therefore, pro-PO is involved in both cellular and humoral
defense. Melanization begins after recognition and phagocytosis
of the pathogen, when soluble PRRs coupled to target molecules
on bacterial surfaces trigger the serine protease cascade that
results in the cleavage of pro-PO to PO. The PO catalyzes the oxi-
dation of phenols to quinones, which spontaneously polymerize
to form melanin around invading pathogens (Eleftherianos and
Revenis 2011). As overproduction of PO can lead to the produc-
tion of cell-damaging reactive oxygen species, PO activation is
highly controlled by protease inhibitors (Lu et al. 2014).
The form and speed with which melanization occurs vary
according to the virulence of the pathogen and inoculum size
(Pereira et al. 2015; Guerrieri et al. 2019). Typically, melanization
starts with distinct black spots on the surface of the larval cuti-
cle and, as the infection progresses, the larva can become com-
pletely melanized. This event correlates with subsequent larval
death (Tsai, Loh and Proft 2016). After infection, a more pro-
nounced melanization is observed in the dorsal region of the
larva, comprising the heart (Pereira et al. 2015). This region was
identified as the larva’s new immune tissue, because it is flanked
by sessile hemocytes that sequester the pathogens in flux in
the hemolymph, and recruit circulating hemocytes to the heart
region, where they bind to the cardiac muscle and continue to
phagocytose pathogens (King and Hillyer 2012; Hillyer 2016). Our
previous results showed how G. mellonella’s cellular and humoral
immune responses are integrated: the site with the strongest
melanization (the heart) is also the region with the highest con-
centration of hemocytes (Pereira et al. 2015).
Although innate immune responses are non-specific, they
are the first line of defense against invading pathogens, being
widely distributed throughout the body to maintain homeosta-
sis and prevent infections (Sheehan et al. 2018). The lack of
an adaptive immunity in G. mellonella and other insects can be
seen as an advantage for research purposes, since the model
allows the study of host–pathogen interactions and related
innate immunity mechanisms without the interference of adap-
tive responses (Kavanagh and Sheehan 2018). Therefore, the
G. mellonella model can substitute for more complex vertebrate
models, depending on the objective of the study, and when the
absence of the adaptive immune system limits the possibility of
replacing vertebrate models, G. mellonella can be an intermediate
model between in vitro and vertebrate in vivo studies.
USING G. MELLONELLA TO INVESTIGATE THE
VIRULENCE OF BACTERIAL PATHOGENS
The potential of a bacterium to cause disease is related to its
repertoire of virulence factors. In general, virulence factors are
divided into those that: (i) allow entry and establishment of
infection in the host; (ii) contribute to the evasion of the host’s
immune response; and (iii) allow the bacteria to multiply and
spread throughout the host or to a new host. However, bacterial
virulence is complex and multifaceted. Species and strains have
different sets of virulence genes, and these can be subjected to
regulation, which makes virulence even more diverse (Diard and
Hardt 2017; Weigel and Dersch 2018).
In this context, G. mellonella has been extensively used to
study bacterial virulence (Fig. 4), which can be evaluated in sev-
eral ways after inoculation with a pathogenic microorganism,
such as observing melanization and larval death over time (Kay

Figure 4. Use of G. mellonella model to study bacterial virulence. The graphics show the diversity and representativeness of Gram-negative and Gram-positive species
that have had their virulence investigated by the G. mellonella model. The data were obtained after a bibliographic search conducted in July 2020 at PubMed (MEDLINE
database) with the keyword ‘Galleria mellonella’, and subsequent individual assessment of the nature of each scientific article.
et al. 2019), density and morphology of hemocytes (Pereira et al.
2015; Sheehan et al. 2019), expression of enzymes and AMPs
(Moghaddam et al. 2016) and histopathological effects (Meir et al.
2018).
Below, we discuss established virulence factors of represen-
tative major human and veterinary bacterial pathogens, and
relevant virulent studies in G. mellonella infection models. The
information in the text is also summarized and supplemented
with the findings for other relevant and well-studied pathogens
within the ESKAPE group (comprising Enterococcus faecium, Stap
hylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa, Enterobacter spp.), in Table 1.
Galleria mellonella studies on the virulence of S. aureus
Staphylococcus aureus is the most widely studied Gram-positive
pathogen in G. mellonella infection models (Fig. 4). Given its
prevalence and the increasing level of strains resistant to multi-
ple drugs, S. aureus is classified by the World Health Organization
(2017) as one of the priority microorganisms in studies for new
antimicrobial therapies. First, we will discuss some major viru-
lence factors of S. aureus, and then some studies in G. mellonella
investigating S. aureus pathogenicity.
Staphylococcus aureus has a broad repertoire of virulence
genes, enabling the bacterium to cause several types of diseases,
including toxic shock syndrome, sepsis, endocarditis, pneumo-
nia, food poisoning, and skin and soft tissue infections (Lee et al.
2018). Among the most studied virulence factors of this bac-
terium are surface-associated and secreted proteins (Laabei et al.
2015; Tam and Torres 2019). The collectively called ‘microbial
surface components recognizing adhesive matrix molecules’
(MSCRAMMs) are proteins that mediate the adherence of S.
aureus to host tissues, including clumping factors A and B (ClfA
and ClfB), fibronectin-binding proteins (FnBPs), collagen-binding
adhesin (Cna) and elastin-binding protein (Ebps). Staphylococ-
cus aureus also maintains its cellular aggregation, including in
Pereira et al. 5
biofilms, by expressing the intercellular polysaccharide adhesin
molecule (PIA) encoded by the ica operon (Haddad et al. 2018; Ma
et al. 2019).
Some staphylococcal toxins weaken the host’s response, as
they degrade host cells, and manipulate both innate and adap-
tive immune responses, and contribute to S. aureus proliferation
(Tam and Torres 2019). Among the main toxins of S. aureus are
pore-forming toxins, such as hemolysin, and leukotoxins, e.g.
the Panton–Valentine - PVL (Oliveira, Borges and Simões 2018;
Tam and Torres 2019). Staphylococcus aureus also produces a vari-
ety of virulence factors with enzymatic properties. They can
break down host barriers or molecules for nutrient acquisition,
bacterial survival and dissemination (e.g. coagulase, staphylok-
inase, nucleases, proteases, metalloprotease, serine proteases,
cysteine proteases, hyaluronidase and lipases) (Tam and Torres
2019).
Staphylococcus aureus has a highly clonal population struc-
ture with clonal complexes (CCs) comprising closely related,
although not identical, genetic backgrounds (Dayan et al. 2016;
Turner et al. 2019). Pérez-Montarelo et al. (2017) analyzed the vir-
ulence of S. aureus strains from five representative CCs associ-
ated with endovascular complications, using the G. mellonella
model. This was the first study comparing the virulence of dif-
ferent S. aureus CCs in vivo, and revealed significant differences
between them. CC30 strains exhibited low virulence against
G. mellonella, corroborating previous studies that had reported
significant virulence attenuation of CC30 in G. mellonella and
murine sepsis models (Sharma-Kuinkel et al. 2017), while CC15
strains displayed high virulence. Genetic analysis of CC15 strains
revealed that they had specific virulence gene variants, which
were likely associated with the phenotype of higher virulence
against G. mellonella. These CC15 virulence variants were more
notable in genes encoding for leukocidins and proteases. One
CC15 strain presented a particularly high virulence phenotype,
which was correlated with the presence of the lukFS genes, cod-
ing for PVL.
 6 Pathogens and Disease, 2020, Vol. 00, No. 00

Table 1. Overview of some studies to assess several aspects of bacterial virulence and treatment with antimicrobial compounds, with emphasis
on species of the ESKAPE group and Escherichia coli.

Bacterial species Type of study Reference

Acinetobacter Comparison of virulence of different strains (Chusri et al. 2019)

baumannii
Study of mutant strains for important virulence factors (Weidensdorfer et al. 2019;
Niu et al. 2020)
Discovery of a new hypervirulent strain (Zhou et al. 2020)
Combination of drugs with adjuvants to treat infections caused by MDR strains to (Minrovic et al. 2018)
reduce colistin dose
Antimicrobial peptide therapies for the treatment of infections caused by MDR (Chung et al. 2016)
strains
Plant natural compounds to control bacterial infections caused by MDR strains (Betts et al. 2017)
Enterobacter sp. Comparison of virulence of different subpopulations of clinical isolates (Brust et al. 2019)
Characterization of virulence of a representative strain of a new species of the (Pati et al. 2018)
genus Enterobacter
Combination of therapies to treat infections caused by MDR strains (Betts et al. 2014; Yang et al.
2016)
Control of infections with bacteriophage therapy (Manohar, Nachimuthu and
Lopes 2018)
Enterococcus sp. Comparison of virulence among vancomycin-resistant clinical strains (Lam et al. 2013)
Study of mutant strains for important virulence factors (Salze et al. 2020; Zheng et al.
2020)
Antimicrobial photodynamic therapy for the treatment of infections caused by (Chibebe et al. 2013)
vancomycin-resistant strains
Combination of antimicrobial therapies to treat infections (Thieme et al. 2020)
Escherichia coli Understanding of regulatory networks that modulate the expression of LEE (Morgan, Ortiz and Riordan
virulence factors in EHEC with studies of gene expression and mutant virulence 2014; De la Cruz et al. 2016)
Comparison of virulence among clinical strains of typical and atypical EAEC (Guerrieri et al. 2019)
Assessment of regulation mechanisms of innate immunity against infections (Mukherjee et al. 2020)
caused by UPEC strains by miRNAs
Combination of antimicrobial therapies to treat infections caused by MDR and (Brochado et al. 2018)
XDR strains
Antimicrobial peptide therapies for the treatment of infections caused by MDR (Vergis et al. 2019)
strains
Control of infections with bacteriophage therapy (Manohar, Nachimuthu and
Lopes 2018)
Klebsiella pneumoniae Study of mutant strains for important virulence factors (Insua et al. 2013; Mills et al.
2017)
Discrimination of strains regarded as highly virulent from less virulent strains (Insua et al. 2013)
Comparison of virulence of colistin-resistant isolates (Esposito et al. 2018)
Discrimination of virulence level of hvKP strains (Shi et al. 2018; Li et al. 2020)
Combination of therapies to treat infections caused by KPC strains (Nath et al. 2018)
Control of infections with bacteriophage therapy (Manohar, Nachimuthu and
Lopes 2018; Thiry et al. 2019)
Staphylococcus Comparison of virulence of different clonal complexes (CCs) of clinical relevance (Pérez-Montarelo et al. 2017)
aureus
Assessment of the relationship between the extracellular proteome and the (Zhao et al. 2019)
virulence of strains
Characterization of infection development (Sheehan, Dixon and
Kavanagh 2019)
Combination of antimicrobial therapies to treat infections (Li et al. 2019)
Antimicrobial peptide therapies to treat infections caused by MRSA strains (Yuan et al. 2019)
Use of plant natural compounds to control bacterial infections caused by MDR (Knidel et al. 2019; Bezerra
strains Filho et al. 2020)
Control of infections with bacteriophage therapy (Tkhilaishvili et al. 2020)
Pseudomonas Comparison of virulence of different strains (Medina-Rojas et al. 2020)
aeruginosa
Study of mutant strains for important virulence factors (Klein et al. 2019; Lo Sciuto
et al. 2019)
Screening for virulence of new strains (Kaszab et al. 2019)
Combination of drugs with adjuvants to treat infections caused by MDR and XDR (Brochado et al. 2018; Idowu
strains et al. 2019)
Antimicrobial peptide therapies for the treatment of infections caused by MDR (Zheng et al. 2017)
strains
Control of infections with bacteriophage therapy (Forti et al. 2018)
Actinobacillus Discriminate the virulence of different isolates (Pereira et al. 2018)
pleuropneumoniae
Study of mutant strains for a pleiotropic regulator (Pereira et al. 2015)

Another study, carried out by Graf et al. (2019), evaluated in
G. mellonella the activity of virulence factors from the extracel-
lular matrix of biofilms of S. aureus, an essential determinant of
the colonization of medical devices that make this bacterium
one of the major causes of nosocomial infections. These fac-
tors were identified by a multiomic approach and included cap-
sule proteins, hemolysins, leukotoxins and lipases. The cell-free
biofilm supernatant containing these factors was injected into
the hemolymph of G. mellonella larvae, leading to a high mortal-
ity rate and demonstrating the utility of the model in the study
of virulence factors secreted by S. aureus.
The extracellular proteome (exoproteome) of S. aureus is a
main reservoir of virulence factors, showing substantial hetero-
geneity for different clonal lineages (Ziebandt et al. 2010; Zhao
et al. 2019). A study by Zhao et al. (2019) showed that the vir-
ulence of S. aureus spa-type t437 may be directly related to its
exoproteome profile, and that the G. mellonella killing assay can
accurately differentiate between these profiles. Certain proteins
such as IsaA, IsdB, IsdA, IsdE, IsdH and Chitinase B were related
to the killing of larvae. The IsaA protein, a major antigen of S.
aureus, was exclusive to the exoproteome of groups that were
highly virulent against G. mellonella, in line with the attenuated
activity of an isaA deletion mutant. Chitinase B was also exclu-
sive to the exoproteome of the highly virulent groups, suggesting
that this enzyme may impact immune evasion and infection in
G. mellonella, similar to its activity in human hosts (da Silva et al.
2009). Mutants for isdA or isdB were not attenuated in G. mel-
lonella, showing that these siderophores may not be relevant to
cause infection in this insect.
The use of G. mellonella for studying S. aureus is endorsed by
the strong interaction of this pathogen with the humoral and
cellular immune response of this insect. Staphylococcus aureus
induces the expression of a variety of peptides related to the
immune system and also forms nodules, which are similar in
structure and function to abscesses commonly found during
S. aureus skin and soft tissue infections in humans (Sheehan,
Dixon and Kavanagh 2019). This shows that S. aureus virulence
factors are recognized by the larval immune system, and its
elimination occurs through mechanisms similar to those of its
natural (human) host.
It is noteworthy that the most common, accurate and reliable
way to assess the virulence of bacteria in G. mellonella is through
the inoculation of the microorganism directly in the insect’s
hemolymph. This practice, however, saves the pathogen from
obstacles that are usually present in the initial stages of infec-
tion, that are overcome by factors involved in adherence, break-
ing barrier integrity and colonization. Since these obstacles are
absent in G. mellonella assays, the bacteria are readily confronted
by the larval innate immune system’s components. Therefore,
studies on virulence factors involved in the early stages of infec-
tion, as well as those factors that have very specific targets, need
special attention during experimentation, so the results can be
properly interpreted.
Most G. mellonella studies with bacterial pathogens focus
on Gram-negative bacteria (Fig. 4). Next, we discuss the recent
advances in understanding the virulence of two of these
microorganisms: E. coli and K. pneumoniae. Both are Enterobac-
teriaceae with high levels of resistance, among the most impor-
tant opportunistic human pathogens and are also classified by
the World Health Organization (2017) as top priorities in stud-
ies aiming at new antimicrobial therapies. Finally, we present
the main findings obtained by our group when studying the vet-
erinary pathogen Actinobacillus pleuropneumoniae, the causative
Pereira et al. 7
agent of swine pleuropneumonia, a contagious disease associ-
ated with high morbidity and mortality in swine herds.
Galleria mellonella studies on the virulence of E. coli
The diversity of E. coli is well documented in terms of its genet-
ics and ability to live as a commensal or pathogen in different
animal and human hosts. This wide diversity arises, in part,
because of constant changes that occur in the E. coli genome,
due to a continuous flow of genetic insertions, deletions and
horizontal gene transfer. While the E. coli genome encodes
∼4000–6500 proteins, the core genome of the species comprises
only 2000 genes. The additional genes determine commensal-
ism, species specificity, pathotypes and antibiotic susceptibility
(Croxen and Finlay 2010; Poolman 2017).
Pathogenic E. coli strains can be divided in two groups: (i)
strains that cause diarrheal intestinal disease (Enteropathogenic
E. coli [EPEC], Enterotoxigenic E. coli [ETEC], Enterohemorrhagic E.
coli [EHEC], Enteroaggregative E. coli [EAEC], Enteroinvasive E. coli
[EIEC] and Diffusely Adherent E. coli [DAEC]), and (ii) strains that
usually cause disease outside the intestinal tract (Extraintesti-
nal Pathogenic E. coli [ExPEC]), being Uropathogenic E. coli (UPEC)
and Neonatal Meningitis E. coli (NMEC), the most common ExPEC
(Croxen and Finlay 2010; Poolman 2017).
The pathogenic strains of E. coli have a wide arsenal of vir-
ulence strategies; many are pathotype specific, but some are
required for virtually all of them. Adhesion to host cells, fre-
quently achieved by fimbriae or pili, is required by all patho-
types, except EIEC. After adhesion, E. coli subverts host cell pro-
cesses, often using secreted proteins and manipulates host cell
signaling pathways, resulting in coordinated host cell invasion,
evasion of host immune responses, efficient colonization and
disease (Croxen and Finlay 2010; Khairy et al. 2019). Seeking to
investigate E. coli virulence, G. mellonella has been widely used
for studying commensal strains (Mukherjee et al. 2020), as well
as those related to diarrheal intestinal disease (De la Cruz et al.
2016; Guerrieri et al. 2019), and those responsible for extraintesti-
nal infections (Williamson et al. 2014; Mukherjee et al. 2020).
Human infections with EHEC can induce hemorrhagic coli-
tis (bloody diarrhea) and the life-threatening hemolytic uremic
syndrome, as a result of the production of Shiga toxin (Stx), the
main virulence factor of EHEC. There are two subgroups of Stx
(Stx1 and Stx2), found in various combinations (Poolman 2017).
EHEC pathogenicity is also involved with its ability to colonize
the gut mucosa, leading to the development of intestinal attach-
ing and effacing (AE) lesions (Nataro and Kaper 1998). Most pro-
teins required for the formation of AE lesions are encoded in a
pathogenicity island called locus of enterocyte effacement (LEE),
which encodes a type III secretion system (T3SS) and numerous
effector proteins (De la Cruz et al. 2016; Schwidder, Heinisch and
Schmidt 2019).
Studies on gene expression and virulence of mutants against
G. mellonella have helped understanding regulatory networks
that modulate the expression of LEE virulence factors. De la Cruz
et al. (2016) showed that the CpxA sensor kinase, which con-
trols E. coli envelope production, is related to the complex reg-
ulatory network of the LEE. This was concluded after the EHEC
cpxA mutant showed reduced expression of LEE genes and was
significantly deficient in killing G. mellonella. The role of TolA in
EHEC pathogenesis and regulation of virulence genes contained
in LEE (i.e. T3SS, common pilus [ecpA] and motility [fliC]) was ver-
ified by combining studies of gene expression and investigation
of tolA mutants, which were substantially attenuated in G. mel-
lonella (Morgan, Ortiz and Riordan 2014).
 8 Pathogens and Disease, 2020, Vol. 00, No. 00
EAEC is another type of pathogenic E. coli related to diar-
rheal intestinal disease and insights into its virulence have been
obtained with the use of G. mellonella (Jønsson et al. 2017; Guer-
rieri et al. 2019). This bacterium is the leading cause of acute
and persistent diarrhea in all ages worldwide (Okhuysen and
DuPont 2010; Spano et al. 2017). Although the pathogenesis of
EAEC is not clear due to the heterogeneity of strains, it is sug-
gested that, after infection, there is an initial adhesion to the
intestinal mucosa, biofilm formation, induction of inflammatory
responses and release of toxins (Okeke et al. 2000; Jensen et al.
2014). The presence of the AggR regulon, a regulator of chro-
mosomal and plasmid-encoded virulence factors, distinguishes
typical from atypical EAEC. It has been suggested that typical
EAECs are more virulent than atypical EAECs (Sarantuya et al.
2004; Morin et al. 2013). In vivo tests with G. mellonella were made
by Guerrieri et al. (2019), indicating that the virulence of EAEC
strains seems to be related, in part, to the AggR regulon, and
atypical EAEC strains can be as virulent as typical strains.
Studies with G. mellonella have also contributed to a bet-
ter understanding of ExPEC virulence. Although they are more
commonly associated with urinary tract infections, ExPEC
strains cause a range of serious infections, including meningi-
tis, pneumonia and bacteremia. These bacteria have an array
of virulence-associated genes (e.g. adhesins, invasins, toxins,
siderophores and polysaccharide capsules), often encoded by
pathogenicity islands (Poolman 2017; Klein and Hultgren 2020).
Williamson et al. (2014) validated the G. mellonella model for
studying ExPEC strains in an analysis that observed that iso-
lates with higher amounts of virulence genes killed larvae sig-
nificantly faster than isolates with lower virulence genes scores.
In contrast, no relationship was found between the virulence
factor (VF) score and LD50 for ExPEC strains associated with ade-
nomatous polyps (Ambrosi et al. 2019). Therefore, a high VF score
does not necessarily correlate with larval mortality. The results
suggested that the presence of other genes, either not investi-
gated or unknown, or even regulatory mechanisms, may be nec-
essary for the complete expression of virulence genes.
Studies of G. mellonella infection with UPEC led to the discov-
ery of new miRNAs involved with the epigenetic reprograming of
the larval innate immunity. These miRNAs were not expressed
when the infection was carried out with commensal strains,
suggesting a novel therapeutic application to treat urinary tract
infections in humans (Mukherjee et al. 2020).
Galleria mellonella studies on the virulence of K.
pneumoniae
Klebsiella pneumoniae is another increasingly problematic oppor-
tunistic pathogen, mainly due to its high rates of antibiotic resis-
tance, and difficulty in treatment. The emergence and spread
of multidrug-resistant (MDR) K. pneumoniae is a serious public
health threat, which is aggravated by the increased prevalence
of colistin resistance (Pitout, Nordmann and Poirel 2015; Pet-
rosillo, Taglietti and Granata 2019). Although the mechanisms
of K. pneumoniae antimicrobial resistance are widely studied,
its virulence mechanisms are still poorly understood, with the
capsule, fimbriae, LPS and siderophores (i.e. enterobactin, aer-
obactin, salmoquelin and yersiniabactin) being the most studied
(Wand et al. 2013; Mills et al. 2017).
Klebsiella pneumoniae strains are divided into classical (cKP)
and hypervirulent (hvKP). Currently, most K. pneumoniae infec-
tions are caused by cKP strains, however, the incidence of infec-
tions due to hvKP has been steadily increasing over the past
three decades in countries of the Asian Pacific Rim (Russo and
Marr 2019). The hvKP strains are more virulent than cKP, which
makes their identification critical for patient care and epidemi-
ologic studies (Li et al. 2020; Russo and Macdonald 2020).
Initially, hvKP strains were correlated with the extreme-
stickiness ‘hypermucoviscous’ phenotype. However, it is now
known that not all hvKP strains are hypermucoviscous, and
some cKP strains have this characteristic, suggesting that hyper-
mucoviscosity and hypervirulence are two different phenotypes
of K. pneumoniae (Catalán-Nájera, Garza-Ramos and Barrios-
Camacho 2017). Although early hvKP isolates were sensitive to
antimicrobials, MDR-hvKP strains have been reported, raising
concerns about treating infections caused by these bacteria (Shi
et al. 2018; Wyres et al. 2019).
Genotypic markers, such as peg-344, iroB, iucA, rmpA and
rmpA2, present on the hvKP virulence plasmid can be used to
identify hvKP; however, some low-virulence strains also carry
these markers (Russo and Marr 2019; Li et al. 2020). Several
investigations have examined whether the K1 and/or K2 cap-
sule types enhanced virulence compared with other types. How-
ever, although it is possible that the capsule type could modu-
late hvKP virulence, hypervirulence is not exclusive to these two
types of capsule (Russo and Marr 2019).
The need for an in-depth investigation of K. pneumoniae viru-
lence is evident, and G. mellonella has been extensively used for
studying cKP (Insua et al. 2013; Mills et al. 2017; Esposito et al.
2018) and hvKP (Shi et al. 2018; Li et al. 2020; Russo and Macdon-
ald 2020).
Insua et al. (2013) showed that G. mellonella could discrim-
inate strains regarded as highly virulent from others less vir-
ulent, in addition to pinpoint differences among highly viru-
lent strains. They also showed that mutants lacking capsule
polysaccharides, lipid A decorations, or the outer membrane
proteins OmpA and OmpK36, previously known to be attenuated
in the mouse pneumonia model, were also attenuated in G. mel-
lonella. This study also showed that G. mellonella infection with
K. pneumoniae might lead to some of the main characteristics
of Klebsiella-induced pneumonia (i.e. cell death associated with
bacterial replication, prevention of phagocytosis and attenua-
tion of host defense responses, mainly the production of antimi-
crobial factors).
A variable virulence profile was also observed by Esposito
et al. (2018), in a study with strains of K. pneumoniae resistant to
colistin, a phenotype mainly regulated by modifications in LPS
structure and ionic charge, which is controlled by the PhoQ/PhoP
two-component system. This study observed a variable viru-
lence profile among the isolates, and showed that the MIC of
colistin was correlated with the values of lethal doses against G.
mellonella.
Klebsiella pneumoniae strains can present a great variability in
virulence and it is difficult to correlate certain genetic charac-
teristics with virulence in vivo (Li et al. 2020). One of the biggest
challenges in studying the virulence of this pathogen is to clearly
define hvKP strains and their level of virulence (Shi et al. 2018; Li
et al. 2020). Many groups have attempted to standardize G. mel-
lonella tests to access hvKP virulence (Li et al. 2020; Russo and
Macdonald 2020). However, not all strains characterized as hvKP
are highly virulent in G. mellonella, and although the presence of
some genes associated with the hypervirulence phenotype do
correlate positively with larval mortality, other virulence factors
(which may target molecules absent in G. mellonella) do not.
Thus, so far, the G. mellonella model alone is not capable of
accurately differentiating strains of hvKP and cKP, a possible

consequence of the complex genetic background of clinical iso-
lates (Shi et al. 2018; Li et al. 2020). The fact that some cKP strains
are highly virulent in G. mellonella indicates that the larva is sen-
sitive to some virulence factors that are still unknown, and fur-
ther investigation is needed to discover these factors (Li et al.
2020). Even so, the model has proved to be an excellent tool for
the study of mutant strains of K. pneumoniae. The advance of
genomic studies and functional genetic analysis will hopefully
improve our understanding of K. pneumoniae virulence.
Virulence of A. pleuropneumoniae
Studies performed by our group showed the suitability of the
G. mellonella model to discriminate isolates and different strains
(wild type and mutant) of the pig pathogen A. pleuropneumoniae
presenting varied levels of virulence (Pereira et al. 2015, 2018).
Although the virulence of this pathogen is mostly related to
the production of pore-forming RTX toxins, we observed that
mutants for the chaperone Hfq are attenuated in the model, as
seen in pigs, the natural host. Since Hfq is a global regulator of
the activity of small regulatory RNAs (sRNAs) in this bacterium,
our studies indicate that sRNAs regulate virulence in A. pleurop-
neumoniae, though the mechanisms have yet to be determined
(Pereira et al. 2015; work in progress).
USING G. MELLONELLA TO EVALUATE THE
EFFICACY OF ANTIMICROBIAL THERAPIES
Treating infections caused by MDR bacteria is a major pub-
lic health challenge worldwide. In addition to the increase of
bacterial resistance to currently available antimicrobials, there
has been little progress in the development of new antimicro-
bials, increasingly limiting therapeutic options (Nathan 2020).
The Gram-negative bacteria E. coli, K. pneumoniae, P. aeruginosa,
A. baumannii and Neisseria gonorrhoeae, and the Gram-positive S.
aureus and Enterococcus spp. have all been identified as critical
pathogens with particularly high rates of resistance to antibi-
otics, resulting in a reducing pool of treatments available for
these organisms (CDC 2019). In this context, in addition to study-
ing bacterial virulence, G. mellonella has been extensively used
to evaluate the efficacy of several types of antimicrobial thera-
pies (Fig. 5), against Gram-negative and Gram-positive bacteria
(Fig. 6).
Aiming at overcoming antimicrobial resistance and decreas-
ing the dose of individual drugs, and therapies with combination
of drugs, especially those already used in clinical practice, have
been suggested and evaluated in G. mellonella (Cutuli et al. 2019).
Combination therapies are often recommended to treat infec-
tions caused by K. pneumoniae producing carbapenemase (KPC),
due to limited treatment options. Using G. mellonella, Nath et al.
(2018) observed that the combination of ceftazidime/avibactam
with carbapenem should be considered in the treatment of seri-
ous infections caused by KPC.
Likewise, studies by Li et al. (2019) indicate that the combina-
tion of linezolid and fosfomycin has synergistic effects against
S. aureus in G. mellonella, and suggest that high doses of line-
zolid and fosfomycin may not be necessary. Thieme et al. (2020)
observed, in G. mellonella, the synergism between ampicillin and
ceftriaxone, efficient to treat high E. faecalis inocula. The study
also questions the benefits of the currently used combination of
ampicillin and the nephrotoxic antibiotic gentamicin, instead of
the safer ceftriaxone.
Pereira et al. 9
Combining drugs with adjuvants is another strategy to
enhance the effectiveness of antibiotics and delay the emer-
gence of resistance. Idowu et al. (2019) observed, in G. mellonella,
that the non-ribosomal tobramycin–cyclam conjugate confers
a potent adjuvant property that restores the complete antibi-
otic activity of meropenem and aztreonam against carbapenem-
resistant P. aeruginosa, presenting a way to further preserve the
therapeutic utility of β-lactam antibiotics.
Colistin, one of the last resources to treat Gram-negative MDR
infections, has recognized high renal and neurological toxicity.
In view of the growing need to use this antibiotic, the use of adju-
vants in clinical practice could possibly decrease the dosage of
the medication. In this context, Minrovic et al. (2018) presented
a new urea-containing class of 2-aminoimidazole-based adju-
vants that potentiates colistin activity against A. baumannii. The
adjuvants allowed a 1000-fold reduction in the MIC of colistin in
vitro, and showed efficacy in G. mellonella, representing the first
step to validate the potential of these adjuvants to reduce the
colistin dose.
In a much broader study, a pairwise drug combination
approach was evaluated by Brochado et al. (2018), with dozens
of antibiotics of all major classes, food additives and adjuvants,
to assess the antagonism or synergistic activity of almost 3000
drug combinations against E. coli, Salmonella enterica serovar
Typhimurium and P. aeruginosa. After in vitro experiments, syn-
ergistic combinations were tested in vivo against MDR strains
of these pathogens in the G. mellonella model. In addition to
identifying drug combinations that were effective against these
bacteria, this study observed that synergism is more common
between compounds that target different steps of the same pro-
cess, in a manner that is mostly species specific, revealing a
great potential for narrow-spectrum therapies.
AMPs are another promising source for the development of
therapies against antibiotic resistance. These, used alone or in
combination with antibiotics, have been tested in G. mellonella to
treat infections by A. baumannii, E. coli, P. areuginosa and S. aureus
(Chung et al. 2016; Zheng et al. 2017; Vergis et al. 2019; Yuan et al.
2019).
Yuan et al. (2019), for example, evaluated the antimicrobial
activity of the AMP japonicin-2LF, isolated from frog skin secre-
tion, against bacterial cystic fibrosis isolates. The AMP increased
the survival of G. mellonella infected with MRSA and was shown
to increase the permeability of the bacterial membrane.
Zheng et al. (2017) studied the AMP cecropin A2, produced by
Aedes aegypti, and its activity against P. aeruginosa. In addition
to increasing the survival of G. mellonella larvae infected with
P. aeruginosa when applied alone, cecropin A2 also had a syner-
gistic effect with tetracycline. This synergism was attributed to
the fact that cecropin A2 permeabilizes the bacterial membrane,
facilitating the entrance of tetracycline into the cell.
Plant natural compounds have also been widely tested in G.
mellonella to control bacterial infections. Betts et al. (2017) tested
the efficiency of the natural polyphenols theaflavin and epicat-
echin, alone and in combination, against six clinical isolates
of MDR A. baumannii. The polyphenol combination theaflavin–
epicatechin produced effective antibacterial activity and showed
great potential for the treatment of infections caused by MDR A.
baumannii.
An essential oil isolated from Eugenia brejoensis, a plant from
the Brazilian semiarid ecosystem, has antimicrobial activity.
Bezerra Filho et al. (2020) showed that, in addition to killing sev-
eral MDR S. aureus strains, subinhibitory concentrations of this
oil reduced bacterial hemolytic activity, and increased its sus-
ceptibility to hydrogen peroxide. When applied to G. mellonella
 10 Pathogens and Disease, 2020, Vol. 00, No. 00

Figure 5. Applications of the G. mellonella model to study bacterial pathogens and antimicrobial agents. Depending on the nature of the arsenal of virulence factors of
a given bacterial population, different strains can lead to different patterns of larval melanization and killing (distinct strains are depicted in the top left of the figure
as non-encapsulated cells, encapsulated cells and flagellated cells, respectively); the same applies to the study of mutants for virulence genes (top center). In the face
of the ongoing increase of antimicrobial resistance, G. mellonella has been extensively used to evaluate alternative therapies to combat bacterial infections, including
the study of antimicrobial activity of classical drugs or the combination of drugs (top right); the activity of natural compounds, mostly extracted from plants (bottom
left); the combination of classical drugs with adjuvants (bottom center); and the activity of bacteriophages (phage therapy, bottom right).

Figure 6. Use of G. mellonella model to evaluate antimicrobial therapies. The graphics show the diversity and representativeness of Gram-negative and Gram-positive
species that were used to assess the effectiveness of different antimicrobial therapies in G. mellonella model. The data were obtained after a bibliographic search
conducted in July 2020 at PubMed (MEDLINE database) with the keyword ‘Galleria mellonella’, and subsequent individual assessment of the nature of each scientific
article.

infected with S. aureus, this natural compound increased larval
survival, while decreasing melanization, and bacterial load in
the hemolymph.
In another study with S. aureus, the antimicrobial potential
of the polyphenol epigallocatechin gallate (EGCG) was tested by
Knidel et al. (2019) against clinical strains with different genetic
profiles, level of virulence and antimicrobial susceptibilities.
EGCG significantly reduced the mortality of G. mellonella larvae
infected with S. aureus, thus being a promising substance for the
treatment of infections caused by this agent.
Another effective approach to control bacterial infections
is therapy with bacteriophages. Due to their highly specific
antimicrobial properties, lytic bacteriophages have re-emerged
as a promising tool for the treatment of drug-resistant bacte-
rial infections. Different phages or phage cocktails have been
used, in treatments with single or multiple doses, to success-
fully treat G. mellonella infected with A. baumannii, Enterobacter
cloacae, E. coli, K. pneumonia, P. aeruginosa and S. aureus (Forti et al.
2018; Manohar, Nachimuthu and Lopes 2018; Thiry et al. 2019;
Tkhilaishvili et al. 2020).

Manohar, Nachimuthu and Lopes (2018) evaluated the ther-
apeutic potential of three different phages against strains of
E. coli, K. pneumoniae and E. cloacae using G. mellonella. Larvae
infected with E. coli and E. cloacae had to be treated with three
phage doses at a 6 h interval to achieve a 100% survival rate.
However, in the case of K. pneumoniae, a single phage dose treat-
ment was sufficient. More recently, Thiry et al. (2019) showed the
effectiveness of new bacteriophages against emerging ST23 and
ST258 lineages of K. pneumoniae, reducing the mortality rate of
G. mellonella larvae from 70% to 20%.
Bacteriophage therapy can also be a great strategy for the
treatment of bacterial infections associated with biofilms, such
as those caused by S. aureus. In this sense, Tkhilaishvili et al.
(2020) evaluated the ability of two commercially available Staphy-
lococcal bacteriophages (PYO and Sb) to disrupt biofilms, control
biofilm formation and to control the development of an infec-
tion caused by methicillin-resistant S. aureus (MRSA). In the face
of a systemic infection, both bacteriophages increased the sur-
vival of G. mellonella, by either preventing or treating the infec-
tion, similarly to the effect observed in the treatment with van-
comycin.
The main advantages offered by G. mellonella in the study of
new antimicrobial strategies are: (i) precision of the inoculum
injected directly into the hemocoel, ensuring precise concentra-
tions of the compounds tested; (ii) the relatively large size of the
larvae, which allows more than one inoculation to be carried out
without major physical trauma; and (iii) it facilitates the screen-
ing of a set of drugs, so that a smaller number of compounds
need to be evaluated in additional tests with vertebrates.
The use of G. mellonella to evaluate new antimicrobial strate-
gies is undoubtedly very important for rapid identification of
novel lead compounds, and new therapeutic strategies. How-
ever, there are considerations required when carrying out such
experiments in G. mellonella. The tests must be carried out with
bacterial doses that do not kill the larva quickly, and the tox-
icity of the compound used must also be tested. Experiments
must be organized and precisely timed so that there are no vari-
ations in treatment times, especially in experiments with many
treatments. Much attention should also be paid to the inocula-
tion site; to avoid further trauma, it is suggested to alternate the
inoculation sides.
PRACTICAL CONSIDERATIONS ON THE USE OF
THE G. MELLONELLA IN RESEARCH WITH
BACTERIAL PATHOGENS
Given what has been discussed so far, the various applications
and advantages offered by the G. mellonella model to scientific
research are clear. The model allows advances in research with
a wide diversity of bacterial pathogens, both in aspects related to
virulence and to the use of new antimicrobial therapies (Figures
S1 and S2, Supporting Information). However, beyond the critical
sense of the researcher for the interpretation of the data, taking
into account the particularities of the model and the microor-
ganism, the success of the study and consistency of the data
obtained depend on simple factors that are often overlooked.
How is the G. mellonella that you use reared?
One of the greatest difficulties in standardizing experiments
with G. mellonella is related to the origin of the larvae used. Lar-
vae can be reared in research laboratories at low cost, but can
also be easily purchased from outside suppliers. The lack of
Pereira et al. 11
outlets whereby researchers can purchase specific genotypes of
G. mellonella, where bred under standard conditions, is a great
limitation. Currently, there are only a few companies that sup-
ply standardized G. mellonella larvae for scientific purposes (e.g.
BioSystems Technology, UK). Most of the suppliers of G. mel-
lonella used in research are independent breeders specialized in
selling larvae as animal food rather than for use in scientific
research. Therefore, there is no appropriate standardization in
the protocols for rearing the insects. In addition, companies that
supply G. mellonella for non-scientific purposes often increase
productivity by using antibiotics and juvenilizing agents that
alter larval metabolism (Cutuli et al. 2019).
Some laboratories, like ours, rear their own G. mellonella for
scientific research. By doing so, it becomes clearer how impor-
tant it is to standardize the rearing protocol for G. mellonella,
and how sensitive the larvae can be to diet and environmen-
tal variations, such as temperature, exposure to light, physical
stress, humidity and proximity to moths or pupae. In this sense,
Jorjão et al. (2018) tested different artificial diets and observed
several physiological variations in G. mellonella. The study then
suggests a diet composition that benefits the life cycle of G.
mellonella, increasing the larval weight, and also stimulating
their immune system by increasing the hemolymph volume
and hemocyte concentration, generating larvae that are more
resistant to infection by different microorganisms. Therefore, we
emphasize that the standardization of an artificial diet, which
guarantees that G. mellonella are healthy physiologically and in
terms of immune function, is important to minimize external
influences on the results.
Temperature has a great influence on both G. mellonella devel-
opment and immune response (Wojda 2017). Studies show that
the exposure of larvae to heat induces immune responses,
increasing the expression of AMPs, while prolonged exposure
to low temperatures decreases the density of hemocytes and
proteins associated with metabolic pathways and prophenolox-
idase, affecting the larva’s susceptibility to microbial pathogens
(Browne et al. 2015; Wojda 2017).
Therefore, it is evident that standard G. mellonella rearing
protocols, regarding diet, temperature and other factors, facil-
itates reproducibility of results both within and between lab-
oratories. The act of purchasing larvae from non-specialized
companies keeps the researcher from controlling situations that
might influence studies’ outcomes.
Other factors influencing reproducibility
In addition to the aforementioned considerations related to
the rearing of G. mellonella, other factors that influence the
results obtained with infection models are worth mentioning.
Pre-treatments are one of those factors and can drastically alter
the results of a study with G. mellonella.
A widely used pre-treatment is the incubation of the larvae at
low temperatures from one to several days. As previously men-
tioned, this practice reduces the immune response of G. mel-
lonella and thus, the virulence of a microorganism tested can be
overestimated. However, this pre-treatment can be practiced if
the study involves pathogens with relatively low virulence, as
described by Browne et al. (2015).
Another common practice is leaving larvae without food for a
variable period before the experiment (Ramarao, Nielsen-Leroux
and Lereclus 2012; Andrea, Krogfelt and Jenssen 2019). Similar
to incubation at low temperatures, starvation also compromises
the immune response of larvae making them more susceptible
to infection (Banville, Browne and Kavanagh 2012).
 12 Pathogens and Disease, 2020, Vol. 00, No. 00
Other factors that can influence the results are: (i) prepa-
ration of the inoculum (concentration of microorganisms and
growth phase must be well defined); (ii) form of inoculation
(intrahemocoelic injection, the most used, or oral gavage of
gut pathogens); (iii) definition of larval death (some researches
observe melanization, others also observe larval movement
and/or response to touch); and (iv) number of larvae used in the
experiment.
One of the great advantages of using an invertebrate model is
the possibility of working with large numbers of individuals in
parallel, and the possibility of greater statistical validity. Stud-
ies often use from 5 to 15 larvae per group (Pereira et al. 2015;
Li et al. 2020; Russo and Macdonald 2020). Most researchers use
groups of 10 larvae for each treatment, with three experimen-
tal and three biological replicates. Experiments with a smaller
number of larvae might not be reproducible, and generate uncer-
tainties in the interpretation of the results.
The aforementioned experimental variables can lead to con-
flicting results obtained with the same reference strains but car-
ried out by different research laboratories. An example is the
comparison of the results of the study by Olsen et al. (2011) to
that of Loh et al. (2013) with the Streptococcus pyogenes strain
MGAS315. Both infected larvae with 106 CFU of bacteria, but
while the first study observed 45% survival after 24 h and 25%
survival after 96 h, the second study observed a survival of 90%
and 70%, respectively, at the same time points. However, each
group had purchased their larvae from different suppliers and
maintained the insects in different conditions.
Recently, conflicting results have also been obtained by
Jønsson et al. (2017) and Guerrieri et al. (2019) in studies with the
EAEC strain 042. In the first case, 100% of G. mellonella larvae were
killed at 24 h post-infection with an inoculum of 104 CFU/larva,
whereas in the second study only 60% of the larvae were dead
at 24 h post-infection with the same strain at a higher dose (105
CFU/larva). In this case, the difference in the virulence profile of
strain 042 can also be attributed to different G. mellonella geno-
types and rearing protocols, or variations in their experimental
protocols.
CONCLUSION
The numerous advantages of the use of G. mellonella have led
to its widespread use as a model to study microbial pathogens.
However, many variables can affect the outcome of such stud-
ies, and the interpretation of results, e.g. the source of the insect
(purchased or laboratory-reared), and prior pre-treatment (diet,
temperature etc.). Microbial virulence is often complex and mul-
tifactorial, and sometimes G. mellonella may not be an appropri-
ate host for specific bacterial pathogens. Although this does not
invalidate the model, it reinforces that well-established ques-
tions and protocols are pivotal to the success of experimen-
tation with G. mellonella. After a decade-long experience with
the model for the study of several bacterial pathogens, we have
observed that the responses for each microorganism vary, and it
is extremely important that researchers understand the genetic
background and physiology of the microorganism studied, so
that the correct interpretation of the results can be made. How-
ever, it is unquestionable that G. mellonella is a versatile, suitable
and reliable model to conduct studies with bacterial pathogens
and antimicrobial agents.
ACKNOWLEDGMENTS
The authors thank Prof. Paul Richard Langford from the Imperial
College London (UK) for his critical analysis of this review and
valuable suggestions.
SUPPLEMENTARY DATA
Supplementary data are available at FEMSPD online.
FUNDING
The authors thank Conselho Nacional de Desenvolvimento
Cientı́fico e Tecnológico—CNPq (Process n. 432065/2018-0),
Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Supe-
rior/Programa de Excelência Acadêmica—Finance Code 001
(CAPES ProEx grant 23038.002486/2018-26) and Fundação de
Amparo à Pesquisa do Estado de Minas Gerais—FAPEMIG for the
financial support.
Conflict of Interest. None declared.
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