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Citation: Bugyna, L.; Kendra, S.;
Bujdáková, H. Galleria mellonella—A
Model for the Study of aPDT—
Prospects and Drawbacks.
Microorganisms 2023,11, 1455.
https://doi.org/10.3390/
microorganisms11061455
Academic Editors: Giulio Petronio
Petronio, Roberto Di Marco and
Laura Pietrangelo
Received: 30 April 2023
Revised: 29 May 2023
Accepted: 30 May 2023
Published: 31 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
microorganisms
Review
Galleria mellonella—A Model for the Study of aPDT—Prospects
and Drawbacks
Larysa Bugyna , Samuel Kendra and Helena Bujdáková*
Faculty of Natural Sciences, Department of Microbiology and Virology, Comenius University in Bratislava,
Ilkovicova 6, 84215 Bratislava, Slovakia; larysa.bugyna@uniba.sk (L.B.); kendra4@uniba.sk (S.K.)
*Correspondence: helena.bujdakova@uniba.sk; Tel.: +421-2-9014-9436
Abstract:
Galleria mellonella is a promising
in vivo
model insect used for microbiological, medical,
and pharmacological research. It provides a platform for testing the biocompatibility of various
compounds and the kinetics of survival after an infection followed by subsequent treatment, and
for the evaluation of various parameters during treatment, including the host–pathogen interaction.
There are some similarities in the development of pathologies with mammals. However, a limitation is
the lack of adaptive immune response. Antimicrobial photodynamic therapy (aPDT) is an alternative
approach for combating microbial infections, including biofilm-associated ones. aPDT is effective
against Gram-positive and Gram-negative bacteria, viruses, fungi, and parasites, regardless of
whether they are resistant to conventional treatment. The main idea of this comprehensive review
was to collect information on the use of G. mellonella in aPDT. It provides a collection of references
published in the last 10 years from this area of research, complemented by some practical experiences
of the authors of this review. Additionally, the review summarizes in brief information on the G.
mellonella model, its advantages and methods used in the processing of material from these larvae, as
well as basic knowledge of the principles of aPDT.
Keywords:
Galleria mellonella; larvae infection; antimicrobial photodynamic therapy; photosensitizer;
irradiation
1. Introduction
In modern medicine and pharmaceutical research, the selection of the appropriate
choice of
in vivo
model has been critical [
1
–
6
]. Research involving vertebrate animals is
subject to strict rules and introduces a number of ethical problems. The European Science
Foundation promotes the need for an ethical approach to each animal experiment. In 1986,
the Council of Europe and the European Union (EU) issued guidelines and legislation on the
use of animals for scientific purposes. Several organizations have prepared guidelines for
their ethical use, and in many countries this is controlled at the level of national legislative
norms. For EU members, national legislation must meet the requirements of Council
Directive 2010/63/EU of the European Parliament, which has been updated from time to
time [
7
,
8
]. Planning any research that involves animals requires following the rules of the
“3 Rs”—replace, reduce, refine. This means that firstly, if possible, it is necessary to replace
vertebrates with invertebrates; secondly, if this is impossible, it is important to reduce their
use to a minimum; and thirdly, to refine the research in such a way as to minimize the
suffering of vertebrates. At the same time, obtaining reliable results should be ensured [
8
,
9
].
Such standards are not so strict for invertebrates, such as the nematode Caenorhabditis
elegans [
10
,
11
], the fruit fly Drosophila melanogaster [
12
,
13
], zebrafish Danio rerio [
14
,
15
], and
the wax moth larvae of Galleria mellonella. The latter is a universal invertebrate model
suitable for conducting various studies that evaluate many different parameters [
4
,
16
–
22
].
G. mellonella has several advantages over other non-vertebrate models (mentioned in
detail in the next section). Additionally, it has some similarities to mammals in terms of
the development of pathologies with mammals [
2
,
4
,
23
]. G. mellonella larvae have a wide
Microorganisms 2023,11, 1455. https://doi.org/10.3390/microorganisms11061455 https://www.mdpi.com/journal/microorganisms
Microorganisms 2023,11, 1455 2 of 21
applicability for each type of assay, which may inform the prospects for further
in vivo
studies in mammals. However, their limitation is a lack of adaptive immune response,
namely the elevation of antibodies and cytokines and the participation of leukocyte killer
cells and dendritic cells, which makes it difficult to predict the immune response in mam-
mals [
23
]. Another obstacle is the lack of availability of mutant larvae, which makes genetic
studies problematic [
2
]. On the other hand, the inoculation of G. mellonella larvae is rapid,
so results can be obtained within a few days. This model is suitable not only for testing
the biocompatibility of different compounds and the kinetics of survival after an infection
followed by subsequent treatment, but also for the evaluation of different parameters
during treatment, including the host–pathogen interaction [4,16,17,24,25].
Antimicrobial photodynamic inactivation (aPDI) is an alternative strategy for fighting
against microorganisms and their biofilms. This approach is based on the use of a non-toxic
dye-photosensitizer (PS), a source of light with the proper wavelength, and the presence of
oxygen. The optimal interplay of all the above factors results in oxidative stress leading to
the death of target cells [
26
,
27
]. In aPDI, there is no specifically targeted component, but
it causes general damage in the cell. Therefore, it is more difficult for microorganisms to
adapt, or even to develop resistance [28].
The main idea of this comprehensive review was to collect information on the use
of G. mellonella in the study of antimicrobial photodynamic therapy (aPDT). Moreover,
the review summarizes in brief information on the G. mellonella model, its advantages,
and the methods used in the processing of material from these larvae. The information
from published works is complemented by the experimental experiences of the authors of
this review.
2. General Characterization of G. mellonela and Significance for Microbiological Research
The larvae of G. mellonella inhabit honeybee hives and feed on bee honeycombs, where
their subsequent pupation takes place. The duration of the life cycle is 8–12 weeks, in-
cluding 5–6 weeks in the larval stage, and larvae are usually about 3 cm long [
29
–
34
].
The advantages of G. mellonella compared to other invertebrate experimental objects can
be summarized as follows: (a) larger larval dimension, which facilitates experimental
manipulations; (b) ability to actively grow over a wider temperature range (20
◦
C–37
◦
C);
(c) shorter period of data acquisition (several days compared to the weeks of other inverte-
brate experimental objects). In addition, the data acquired on G. mellonella are comparable
to the studies obtained on vertebrate animals [
2
,
35
–
37
] as larvae can be tested at the physi-
ological temperature of vertebrates. This is an important factor that allows the study of
temperature-dependent virulence factors [
24
,
29
,
38
–
43
]. In addition, this invertebrate model
is capable of reproducing the clinical signs observed in human infections [42,44,45].
Additional opportunities were identified after the G. mellonella genome was success-
fully sequenced. The level of homology between G. mellonella and humans, mice, or other
model organisms has been determined [
46
,
47
]. Lange et al. (2018) published the results
from genome sequencing using PacBio’s long-read technology. They showed that the
G. mellonella genome consists of 37 genes coding for 13 proteins, 2 rRNA, and 22 transport
RNA. These results greatly contributed to the wider use of this invertebrate model and the
replacement of vertebrates in biomedical research [48–51].
Previously, there were no standardized larvae of G. mellonella, which was a significant
obstacle to its wider use. For many years, they were only commercially available as food for
reptiles and birds, so they were bred, raised, and kept under various conditions. However,
they are now bred specifically for research without the addition of antibiotics or hormones
to the feed. Their age and weight are also monitored, and the cuticle is disinfected to prevent
infections in control groups [
3
,
52
]. The most common method of infection of G. mellonella
larvae is subcutaneous microinjection. Pathogens can also be administered orally, but
commercially available larvae are in the final stage of maturation before pupation, during
which time they are almost non-feeding. In this regard, oral administration is carried out
using a special probe or at an early stage of maturation [52–54].
Microorganisms 2023,11, 1455 3 of 21
G. mellonella is an excellent experimental model for the preliminary screening of the
toxicity and antimicrobial activity of various compounds and disinfectants [
3
,
5
,
55
–
58
]. For
example, manganese-based compounds [Mn(bpqa-
κ3
N)(CO)
3
]Br, [Mn(bqpa
κ3
N)(CO)
3
]Br,
[Mn(CO)
3
(tqa-
κ3
N)]Br, and [Mn(CO)
3
(tpa-
κ3
N)Br, which damage the integrity of the
bacterial membrane, demonstrated antibacterial properties with no toxicity to G. mel-
lonella [
59
]. Similarly, silver nanoparticles (AgNPs) tested by Thomaz et al. (2020) demon-
strated effective antimicrobial activity against Pseudomonas aeruginosa [
60
]. Larva intra-
hemocoel injections were carried out with the antimicrobial peptide (Naphthalene-2-
ly)-acetyl-diphenylalanine-dilysine-OH (NapFFKK-OH) to test its activity against gram-
positive and gram-negative microorganisms [61].
Due to the increase in resistance to antibiotics, silver impurities are widely used, from
lunch boxes to medical device implants. For example, the effectiveness of silver acetate
against the carbapenem-resistant Acinetobacter baumannii was investigated. Using this
compound, the infection of G. mellonella larvae was under control, leading to significantly
improved survival. This study also demonstrated the selective toxicity of silver acetate to
bacterial pathogens without harmful effects on larvae [
62
]. In another study, the effective-
ness of probiotics was studied. Larvae were pre-inoculated with one of two commonly
used probiotic bacteria, Lactobacillus rhamnosus GG [
63
,
64
] or Clostridium butyricum Miyairi
588 [
63
], and then challenged with Salmonella enterica Typhimurium, enteropathogenic Es-
cherichia coli, or Listeria monocytogenes [
44
,
65
,
66
]. The survival rates were increased in larvae
pre-treated with probiotics compared to the control group inoculated with pathogens alone.
Hematocyte density also increased, indicating that both probiotics evocated an immune
response [
63
]. It was also established that G. mellonella larvae can be used to assess the
virulence of anaerobic bacteria of Clostridium perfringens [
67
,
68
]. The results demonstrated
that C. perfringens infection activated the melanization pathway, leading to melanin deposi-
tion. Another study proved the effectiveness of available antibiotics against the biofilms
of multi-drug-resistant Pseudomonas aeruginosa and Klebsiella pneumoniae strains [
69
]. In
addition, the use of G. mellonella larvae makes it possible to evaluate the antibacterial
efficiency of various plant extracts and their ability to modulate the immune response. For
example, pomegranate glycolic extract was effective against Porphyromonas gingivalis, and
it prolonged larval survival compared to the untreated control [70].
In summary, G. mellonella has been used for testing many infections caused by different
gram-positive and gram-negative bacteria. Among gram-positive microorganisms, Staphylo-
coccus aureus [
71
–
77
], Streptococcus pyogenes [
78
–
80
], Streptococcus pneumoniae [
81
–
83
], Strepto-
coccus mutans [
19
,
20
,
84
–
86
], L. monocytogenes [
4
,
44
,
65
,
66
,
87
,
88
], Enterococus faecalis [
89
–
92
],
Enterococcus faecium [
93
–
96
], L. rhamnosus GG [
63
,
64
], C. butyricum Miyairi 588 [
63
], C. perfrin-
gens [
67
,
68
], Mycobacterium bovis [
23
], Mycobacterium abscessus [
97
,
98
], and Mycobacterium
tuberculosis [
99
–
102
] were mentioned. Among gram-negative bacteria, E. coli [
103
–
106
],
S. enterica Typhimurium [
107
–
109
], K. pneumonia [
110
–
113
], A. baumanii [
39
,
114
–
117
], Fran-
cisella tularensis [
118
–
120
], P. aeruginosa [
60
,
121
,
122
], and P. gingivalis [
70
,
123
,
124
] have been
involved. G. mellonella was also used for testing representatives of fungal pathogens, such
as Candida albicans [
21
,
25
,
125
–
128
], Candida dubliniensis [
21
,
25
,
129
,
130
], Aspergillus fumiga-
tus [
38
,
131
,
132
], Cryptococcus neoformans [
57
,
133
–
135
],and Madurella mycetomatis [
45
,
136
–
138
],
and also viruses [139–142] and bacteriophages [143–150].
It has already been mentioned that G. mellonella larvae are a suitable
in vivo
model for
studies related to drug safety and efficacy. Additionally, they can be used for the study of
host–pathogen interactions [
2
,
151
,
152
]. An advantage is the very good survival of G. mel-
lonella at the temperature of the human body, and that they often exhibit symptoms of the
pathogenesis of various diseases similar to those manifested in humans [
4
,
79
,
97
,
153
–
155
].
For example, larvae infected with streptococci manifested clear signs of invasive infection.
Specifically, these included melanization and the formation of a destructive abscess-like
lesion at the inoculation site. These abscesses consisted of dense necrotic tissue in the center
and microorganisms. They were surrounded by a band of host hemocytes, coagulated
hemolymph, and the extracellular pigment melanin. According to the authors, these fea-
Microorganisms 2023,11, 1455 4 of 21
tures are similar to the histopathology commonly seen in mouse and monkey models and
could also be compared with severe soft-tissue infections observed in humans [76].
The protection of G. mellonella from microbial infection has been under intensive study,
and there are some similarities with humans. While the cuticle mimics the skin
[151–154]
,
the immune response mechanism shows signs related to the innate immunity of verte-
brates [
156
–
159
]. Hemocoel contains hemocytes with a similar function to human neu-
trophils that participate in phagocytosis, during which reactive oxygen species (ROS) are
generated [
160
–
163
]. The G. mellonella larvae enable the study of the influence of infection
on the development of oxidative stress and the antioxidant defense system. Additionally, it
has been observed that apoptosis can be initiated during infection [
164
]. Insect hemocyte
extracellular traps (IHETs) were recently described. IHETs act via hemolymph coagulation
and melanization, which contributes to the immobilization and killing of bacteria. These
processes are mediated by a significant release of hemocytes in G. mellonella [156].
Soluble effector molecules orchestrate the humoral response and include complement-
like proteins, such as melanin, and antimicrobial peptides, such as gallerimycin and
galiomicin, which protect G. mellonella from fungal infection, or cecropin, which proved to
be effective during infection with Mycobacterium bovis BCG lux [21,23,46,165–167].
Several approaches are currently available, and these have been adapted and opti-
mized to study various processes in G. mellonella. This analysis could be summarized
into two main areas of study: (i) the kinetics of survival after the testing of an infec-
tion/treatment; (ii) the host–pathogen interaction. The latter scope includes quantitative
analysis, but also qualitative ones that consider hemolymph analysis. Both areas can also in-
volve some molecular biology approaches. Some procedures optimized for the G. mellonella
study are briefly mentioned below with the relevant references.
To determine the progress of infection and the effectiveness of treatment, counting
the number of dead larvae, progress in melanization, the direct counting of pathogens
in body tissues, and histology could be options for evaluation. When determining the
host–pathogen interaction, the most frequent approach is a quantitative analysis providing
information on the number of hemocytes in the hemolymph, the results of which can
give an idea of the level of the immune response [
168
–
171
]. For this purpose, counting the
density of hemocytes, recalculated per 1 mL of hemolymph, can also be conducted [
172
,
173
].
The viability of hemocytes can be investigated using an MTT colorimetric assay. [
174
]. To
characterize the different types of hemocytes, light or phase-contrast microscopy, Giemsa
staining, or neutral red staining is recommended. The enzymatic activity of hemolymph
can be determined by measuring the concentration of insect enzymes involved in immunity,
such as lysozyme and superoxide dismutase [
171
,
175
–
177
]. The label-free quantification
and untargeted analysis of the complete protein profile of hemolymph is usually performed
by proteomic analysis, or proteins can be identified via 2D electrophoresis [
23
,
175
,
178
–
184
].
The implementation of molecular biology is necessary to obtain more detailed informa-
tion from all the above-mentioned aspects of the study of G. mellonella. For the expression
of genes coding for antimicrobial peptides and immunity-related genes, a quantitative
RT-PCR or transcriptomic analysis is optimal [23,175,178,184–186].
The
in vitro
analysis of phagocytosis is performed using fluorescent microscopy of
spotted fluorescent bacteria [
164
,
172
,
174
,
187
,
188
]. Phagocytosis in hemolymph
in vivo
is
analyzed using the same method [
189
]. A detailed study of macrophage activation is
necessary to understand the level of release of ROS or nitrogen species, as well as regula-
tory enzymes [
190
]. Macrophage activation is investigated using the Greiss assay, which
analyzes the release of active nitrogen forms [
37
]. More detailed studies of macrophage
activation are related to the study of DNA damage (ELISA), lipid peroxidation level (mal-
onic aldehyde level), catalase level (fluorometric resorufin assay), or superoxide dismutase
(ELISA) [160,190,191].
In addition to the above-mentioned conventional methods, G. mellonella is a suitable
model for the study of various aspects of aPDT. This issue is addressed in the following
Microorganisms 2023,11, 1455 5 of 21
chapter, including a table summarizing the experimental research over approximately the
last 10 years, since the first work on testing aPDT on G. mellonella was published.
3. Principles of aPDT and the Use of G. mellonella in aPDT
Photodynamic therapy (PDT) was discovered more than a century ago. Its essence
was revealed in detail by Raab, who published a study in 1900 on the use of aPDT as a
cytotoxic technique designed to treat tumors, as well as infectious pathologies [192,193].
The therapeutic effect of PDT (aPDT) is achieved using a photosensitizer (PS) that
is irradiated with light, the emission spectrum of which corresponds to the absorption
spectrum of the PS. In the presence of molecular oxygen, ROS (superoxide, hydroxyl radical,
etc.) and singlet oxygen are generated, which cause the irreversible destruction of a large
range of biomolecules, including nucleic acids, lipids, and proteins [
58
,
93
,
194
]. PDT is
already in use as an alternative approach for the control of malignant diseases. A review
by Ferreira dos Santos et al. (2019) nicely summarized [
179
] the current state of the art
in PDT research and treatment focused on cancer. The authors also introduced in detail
PSs for practical use in the treatment of many cancers. For example, Porfimer, sodium
(Photofrin) was the first PS approved by the Canadian Health Agency in 1993 for the
treatment of bladder cancer. In 1998, the U.S. Food and Drug Administration approved it
for the treatment of early-stage lung cancer. Currently, 11 additional countries in Europe
have accepted the practical use of this PS [
195
]. While the PDT practiced in cancer therapy
is developing dramatically, the application of aPDT for the eradication of pathogenic
microorganisms and viruses is still at a very early stage and has only been developing
faster in the last decade. Nevertheless, several examples of the practical application of
aPDT have already been described, mainly in the treatment of oral diseases. A clinical
study by Fonseca et al. (2022) demonstrated that aPDT reduced the number of infected
anatomical sites in patients with oral candidiasis [
196
]. Another clinical study by Shetty et
al. (2022) proved that a single session of aPDT as an adjunct to mechanical debridement
is effective at reducing peri-implant soft tissue inflammation and oral yeast colonization
in patients with peri-implant mucositis [
197
]. Alves-Silva et al. (2023) used aPDT as an
adjunct to a chemo-mechanical preparation, and it was effective at improving root canal
disinfection and reducing the lipopolysaccharide and lipoteichoic acid levels in teeth with
primary endodontic infection [198].
Generally, aPDI should be an effective method for the eradication of a wide range of
microorganisms, including both gram-positive and gram-negative bacteria, viruses, fungi,
and parasites [
26
–
28
,
199
–
201
]. Due to the fact that PDI is multi-targeted, microorganisms
are not able to develop resistance [
202
,
203
]. Moreover, PDI is highly effective against
microorganisms resistant to conventional antimicrobials [
204
–
208
]. For instance, Štefánek
et al. (2022) used aPDI for the eradication of Candida auris biofilms resistant to antifungal
agents. They found that aPDI significantly decreased the survival of C. auris biofilm
cells, and thus proved to have great potential for the eradication of multi-resistant yeasts.
Furthermore, the observed upregulation of the MDR1 and CDR1 genes did not affect the
overall efficacy of methylene blue-mediated aPDI on biofilms formed by C. auris clinical
isolates, regardless of their sensitivity or resistance [204].
Since aPDT is still under development, optimal models are necessary to investi-
gate not only the effectiveness of treatment after microbial infections, the response of
the immune system, PS cytotoxicity, but also the penetration depth of the light beam.
G. mellonella seems to be an appropriate model for the study of different aspects of aPDT
during infections caused by bacterial and fungal—mono- but also dual or multi-species
biofilms
[18,58,209–212]
. Moreover, PDI can be tested in combination with other bioactive
molecules, including antimicrobial drugs [93,213].
Recently, scientists began to test the effectiveness of aPDT on G. mellonella infected
with C. albicans using different PSs, such as methylene blue [
214
], erythrosine, curcumin, or
toluidine blue [
121
,
209
,
213
]. In the dissertation of Dr. Dadi, the PS phloxine B was tested
Microorganisms 2023,11, 1455 6 of 21
for toxicity in Galleria larvae, and even a 0.5 mM concentration did not exhibit any effect on
G. mellonella survival [215].
The protocol for simple testing is as follows. After the inoculation of the larvae with
a cell suspension of a known density (this should usually be estimated in a preliminary
experiment for each microbial genus or species), the tested PS, diluted in sterile phosphate
buffer saline (PBS) to the desired concentration, is applied to the G.mellonella larvae, usually
by the inoculation method. Using a 10
µ
L Hamilton syringe, 10
µ
L aliquots of the cell
suspension are administrated into the hemocoel of each caterpillar via the proleg at the tail
end of the larva’s body, followed by the administration of the PS via the opposite proleg
(Figure 1) [
58
,
209
,
210
,
212
,
216
,
217
]. For some purposes, the PS can also be applied locally,
as described in a study by Figueiredo-Godoi et al. (2022) [
18
], who used G. mellonella for a
burn model infected with A. baumannii.
Microorganisms 2023, 11, x FOR PEER REVIEW 6 of 21
during infections caused by bacterial and fungal—mono- but also dual or multi-species
biolms [18,58,209–212]. Moreover, PDI can be tested in combination with other bioactive
molecules, including antimicrobial drugs [93,213].
Recently, scientists began to test the eectiveness of aPDT on G. mellonella infected
with C. albicans using dierent PSs, such as methylene blue [214], erythrosine, curcumin,
or toluidine blue [121,209,213]. In the dissertation of Dr. Dadi, the PS phloxine B was tested
for toxicity in Galleria larvae, and even a 0.5 mM concentration did not exhibit any eect
on G. mellonella survival [215].
The protocol for simple testing is as follows. After the inoculation of the larvae with
a cell suspension of a known density (this should usually be estimated in a preliminary
experiment for each microbial genus or species), the tested PS, diluted in sterile phosphate
buer saline (PBS) to the desired concentration, is applied to the G. mellonella larvae, usu-
ally by the inoculation method. Using a 10 µL Hamilton syringe, 10 µL aliquots of the cell
suspension are administrated into the hemocoel of each caterpillar via the proleg at the
tail end of the larva’s body, followed by the administration of the PS via the opposite pro-
leg (Figure 1) [58,209,210,212,216,217]. For some purposes, the PS can also be applied lo-
cally, as described in a study by Figueiredo-Godoi et al. (2022) [18], who used G. mellonella
for a burn model infected with A. baumannii.
Figure 1. Inoculation of G. mellonella larva with 1 mM methylene blue in PBS. (A) An injection station
for the simple manipulation of the larva during inoculation, which involves taping a lter paper
disc to the table and a 1000 µL-disposal tip onto the lter paper. (B) Injection of the larva: the G.
mellonella larva is gently held over the tip using the ngers, or tweezers, with the prolegs at the tail
end of the larva’s body visible. The needle is carefully inserted into a proleg, angling the needle
toward the head of the larva, and 10 µL of methylene blue in PBS is administrated. A dierent proleg
should be used for PS administration than for the previous inoculation of the pathogen to avoid
contamination. (C) During the release of the methylene blue, the larva visibly turns blue.
The application of the PS is followed by irradiation with light of an appropriate wave-
length, and the delivered energy is calculated, taking into account the duration of the ir-
radiation, to determine the total eectivity of irradiation—uence. The PS application
should be approximately 10–30 min before the irradiation, allowing the PS to penetrate
the tissue and nally the microorganisms. During irradiation, energy transfer from the PS
in the presence of oxygen results in the generation of ROS. One molecule of PS can activate
many atoms of activated oxygen. However, it should be considered that the diusion of
the activated oxygen is limited. Another limitation is the proximity to the PS, as objects
distant from it may be subjected to limited or no damage [209,213,218]. The irradiation of
G. mellonella is also a critical step, as it is important to ensure the proper delivery of the
light to cover the desired area of the insect body completely. For this purpose, the larva
should be maintained in a 24-well microtiter plate throughout the irradiation process
Figure 1.
Inoculation of G. mellonella larva with 1 mM methylene blue in PBS. (
A
) An injection station
for the simple manipulation of the larva during inoculation, which involves taping a filter paper disc
to the table and a 1000
µ
L-disposal tip onto the filter paper. (
B
) Injection of the larva: the G. mellonella
larva is gently held over the tip using the fingers, or tweezers, with the prolegs at the tail end of the
larva’s body visible. The needle is carefully inserted into a proleg, angling the needle toward the
head of the larva, and 10
µ
L of methylene blue in PBS is administrated. A different proleg should be
used for PS administration than for the previous inoculation of the pathogen to avoid contamination.
(C) During the release of the methylene blue, the larva visibly turns blue.
The application of the PS is followed by irradiation with light of an appropriate
wavelength, and the delivered energy is calculated, taking into account the duration of the
irradiation, to determine the total effectivity of irradiation—fluence. The PS application
should be approximately 10–30 min before the irradiation, allowing the PS to penetrate the
tissue and finally the microorganisms. During irradiation, energy transfer from the PS in
the presence of oxygen results in the generation of ROS. One molecule of PS can activate
many atoms of activated oxygen. However, it should be considered that the diffusion of
the activated oxygen is limited. Another limitation is the proximity to the PS, as objects
distant from it may be subjected to limited or no damage [
209
,
213
,
218
]. The irradiation
of G. mellonella is also a critical step, as it is important to ensure the proper delivery of
the light to cover the desired area of the insect body completely. For this purpose, the
larva should be maintained in a 24-well microtiter plate throughout the irradiation process
(Figure 2). To prevent the organism from moving around, it is advisable to perform the
irradiation of each larva separately, one by one, and to keep the larva inside its well using
forceps. After performing aPDT, the larvae are incubated in Petri dishes at the required
temperature (usually at 37
◦
C in the dark). Every experiment must include a control group
of G. mellonella larvae that do not receive any injections to monitor the overall quality of the
Microorganisms 2023,11, 1455 7 of 21
larvae over the course of the experiment, as well as a PBS injection control group to ensure
that death was not due to trauma. The survival of aPDT-treated larvae is recorded daily
or hourly, according to a pathological scoring system proposed by Loh et al. (2013) [
78
]
taking into account a few attributes, such as movement activity, melanization, or cocoon
formation. Figure 2illustrates the irradiation of G. mellonella larvae with a red laser, and
how this is processed in the laboratory of Prof. Bujdákováet al.
Microorganisms 2023, 11, x FOR PEER REVIEW 7 of 21
(Figure 2). To prevent the organism from moving around, it is advisable to perform the
irradiation of each larva separately, one by one, and to keep the larva inside its well using
forceps. After performing aPDT, the larvae are incubated in Petri dishes at the required
temperature (usually at 37 °C in the dark). Every experiment must include a control group
of G. mellonella larvae that do not receive any injections to monitor the overall quality of
the larvae over the course of the experiment, as well as a PBS injection control group to
ensure that death was not due to trauma. The survival of aPDT-treated larvae is recorded
daily or hourly, according to a pathological scoring system proposed by Loh et al. (2013)
[78] taking into account a few aributes, such as movement activity, melanization, or co-
coon formation. Figure 2 illustrates the irradiation of G. mellonella larvae with a red laser,
and how this is processed in the laboratory of Prof. Bujdáková et al.
Figure 2. The process of the irradiation of G. mellonella larva with a red laser. (A) PDT assembly; (B)
the G. mellonella larva was positioned in the well of a 24-well microtiter plate; (C) irradiation of the
larva´s body with the red laser (λ = 660 nm, 190 mW/cm2). The distance between the larva and the
laser was 10 cm and the duration of irradiation was 2 min, which corresponded to an energy delivery
of 23 J.
The key factor in PDT (aPDI) eectiveness is PS, which must meet the compatibility
parameters and have high eciency. Absorption in the red and near-infrared spectrum is
also advantageous, as red light is relatively favorable to the treated host. The PS needs to
exhibit only local toxicity, even after light activation. A high level of ROS yield is also
assumed during irradiation [219–221].
Phenothiazinium dyes are the most common PSs used in PDT performed on the G.
mellonella model [58,210,212,216,222]. During the administration of the desired PS into the
larva´s hemocoel, the body of the larva becomes colored, which is an accompanying
Figure 2.
The process of the irradiation of G. mellonella larva with a red laser. (
A
) PDT assembly;
(
B
) the G. mellonella larva was positioned in the well of a 24-well microtiter plate; (
C
) irradiation of
the larva´s body with the red laser (λ= 660 nm, 190 mW/cm2). The distance between the larva and
the laser was 10 cm and the duration of irradiation was 2 min, which corresponded to an energy
delivery of 23 J.
The key factor in PDT (aPDI) effectiveness is PS, which must meet the compatibility
parameters and have high efficiency. Absorption in the red and near-infrared spectrum
is also advantageous, as red light is relatively favorable to the treated host. The PS needs
to exhibit only local toxicity, even after light activation. A high level of ROS yield is also
assumed during irradiation [219–221].
Phenothiazinium dyes are the most common PSs used in PDT performed on the
G. mellonella model [
58
,
210
,
212
,
216
,
222
]. During the administration of the desired PS into
the larva
´
s hemocoel, the body of the larva becomes colored, which is an accompanying
phenomenon. The intensity of the color depends on the concentration of the PS used. Over
the course of the experiment, the larvae excrete the dye and become discolored (Figure 3).
Microorganisms 2023,11, 1455 8 of 21
Microorganisms 2023, 11, x FOR PEER REVIEW 8 of 21
phenomenon. The intensity of the color depends on the concentration of the PS used. Over
the course of the experiment, the larvae excrete the dye and become discolored (Figure 3).
Figure 3. Testing the toxicity of 1 mM methylene blue on G. mellonella larvae. (A) Larvae without an
injection—control group. (B) G. mellonella inoculated with 1 mM methylene blue in PBS. Immedi-
ately after inoculation, the larvae were visibly colored blue. (C) G. mellonella after 24 h. The larvae
slowly began to discolor, and their excrement was blue. (D) G. mellonella after 48 h. The discoloration
was progressing. (E) G. mellonella after 120 h. The larvae were completely discolored and resembled
the control group without any harmful eects.
G. mellonella larvae have been found to be versatile in several studies that evaluated
a PS used in aPDT [210,211,223]. De França et al. (2021) tested in vitro the anti-tumor eect
and skin permeation/retention of a green uorescence pyrene-based dye for aPDT, and
they used the G. mellonella model to determine PS toxicity [224]. Rigoo Caruso et al. (2021)
evaluated the antifungal activity of aPDT in vitro with dierent phenothiazinium PSs
(methylene blue, new methylene blue N, and new methylene blue N Zinc) in combination
with biosynthesized silver nanoparticles. The toxicity of all the tested compounds during
their study was veried in the G. mellonella model [225]. The tests performed in a study by
Malacarne et al. (2023) evaluated the toxicity of porphyrin PS on G. mellonella larvae and
its cytotoxicity on hemocytes. No dark toxicity of PS was observed, even at the highest
concentrations, and even with the longest incubation period (72 h). The intracellular lo-
calization of porphyrin PS was assessed using uorescence microscopy after the hemo-
cytes were isolated and collected from the hemolymph of inoculated larvae [226].
Nowadays, a relatively wide range of PSs are available, including phenothiazine
dyes, porphyrins, chlorines, and phthalocyanines. In addition to synthetic ones, natural
substances such as chlorophyllin, curcumin, and hypericin have also been studied
[18,209,217,227–229].
The development of optimal light sources for PS is important for eective aPDT.
Many PSs used for in vivo testing are activated by a red light with a wavelength between
630 and 700 nm. The source of light is a light-emiing diode (LED light) or diode laser.
The irradiation itself must not aect the survival of the larvae [18,58,212,222,223,230,231].
During the interaction of the tissue with a light beam, most of the light is absorbed,
scaered, or transmied, and only 4–7% is reected. Pigmented tissue areas absorb light
preferentially compared to less pigmented ones [231]. aPDT can also be enhanced by
Figure 3.
Testing the toxicity of 1 mM methylene blue on G. mellonella larvae. (
A
) Larvae without an
injection—control group. (
B
)G. mellonella inoculated with 1 mM methylene blue in PBS. Immediately
after inoculation, the larvae were visibly colored blue. (
C
)G. mellonella after 24 h. The larvae slowly
began to discolor, and their excrement was blue. (
D
)G. mellonella after 48 h. The discoloration was
progressing. (
E
)G. mellonella after 120 h. The larvae were completely discolored and resembled the
control group without any harmful effects.
G. mellonella larvae have been found to be versatile in several studies that evaluated a
PS used in aPDT [
210
,
211
,
223
]. De França et al. (2021) tested
in vitro
the anti-tumor effect
and skin permeation/retention of a green fluorescence pyrene-based dye for aPDT, and
they used the G. mellonella model to determine PS toxicity [
224
]. Rigotto Caruso et al.
(2021) evaluated the antifungal activity of aPDT
in vitro
with different phenothiazinium PSs
(methylene blue, new methylene blue N, and new methylene blue N Zinc) in combination
with biosynthesized silver nanoparticles. The toxicity of all the tested compounds during
their study was verified in the G. mellonella model [
225
]. The tests performed in a study by
Malacarne et al. (2023) evaluated the toxicity of porphyrin PS on G. mellonella larvae and
its cytotoxicity on hemocytes. No dark toxicity of PS was observed, even at the highest
concentrations, and even with the longest incubation period (72 h). The intracellular local-
ization of porphyrin PS was assessed using fluorescence microscopy after the hemocytes
were isolated and collected from the hemolymph of inoculated larvae [226].
Nowadays, a relatively wide range of PSs are available, including phenothiazine dyes,
porphyrins, chlorines, and phthalocyanines. In addition to synthetic ones, natural substances
such as chlorophyllin, curcumin, and hypericin have also been studied [18,209,217,227–229].
The development of optimal light sources for PS is important for effective aPDT. Many
PSs used for
in vivo
testing are activated by a red light with a wavelength between 630
and 700 nm. The source of light is a light-emitting diode (LED light) or diode laser. The
irradiation itself must not affect the survival of the larvae [18,58,212,222,223,230,231].
During the interaction of the tissue with a light beam, most of the light is absorbed,
scattered, or transmitted, and only 4–7% is reflected. Pigmented tissue areas absorb light
preferentially compared to less pigmented ones [
231
]. aPDT can also be enhanced by
increasing the PS concentration. However, higher concentrations of PS can result in the
formation of aggregates, leading to an optical shielding phenomenon that can reduce the
killing of microbial cells [232,233].
Microorganisms 2023,11, 1455 9 of 21
Merigo et al. (2017) studied the use of different laser energy densities (650 nm, 450 nm,
and 532 nm) with or without different types of PSs (toluidine blue, curcumin, and erythro-
sine) in C. albicans infections. The authors suggested that laser irradiation in combination
with an appropriate PS, and even the use of laser irradiation alone, were shown to be
effective at controlling candidiasis using the G. mellonella model [209].
In a study by Figueiredo-Godoi et al. (2019), red laser penetration, delivered at
different fluencies (660 nm, 6 and 15 J/cm
2
), and the distribution of light in the tissue of
G. mellonella larvae was investigated using a power meter and CCD camera. The images
were analyzed according to the interactive 3D Surface Plot plugin of the Image J program.
Subsequently, the concentration of the PS—methylene blue (100
µ
M) which allowed the
best light distribution over the thickness of the larvae’s body after administration was
chosen for the aPDT assays. The authors observed that without the PS, the beginning of
the light distribution in the cuticle occurred at 0.36 mm, and remained for up to 2.5mm. In
association with 100
µ
M methylene blue, the light distribution occurred at 0.27 mm and
extended up to 2.45 mm below the cuticle. These findings suggested that laser irradiation
in association with the proper PS can enhance light distribution in the cuticle [58].
Bispo et al. (2020) performed bacteria-targeted aPDT, which relied on the combination
of a bacteria-specific targeting agent and the light-induced generation of ROS by an ap-
propriate PS in G. mellonella. They conjugated the near-infrared PS IRDye700DX to a fully
human monoclonal antibody, specific to the immunodominant staphylococcal antigen A
(IsaA), creating a novel photo-immunoconjugate. They proved that aPDT with 1D9-700DX
was highly effective at treating G.mellonella infected with a methicillin-resistant strain.
Despite the observed relapse in the bacterial burden 48 h after aPDT, this relapse was not
lethal to the larvae, as there were increased survival rates (~80%) 72 h after treatment.
The authors suggested that the increased survival could be attributed to the innate larval
immune defenses. The authors concluded that aPDT with 1D9-700DX reduced the bacterial
burden to such an extent that the host’s immune responses could overcome infections
caused by multidrug-resistant S.aureus [234].
Chibebe et al. (2013) used G. mellonella for testing the effectiveness of aPDT in the
presence of methylene blue. They demonstrated the prolonged survival of G. mellonella after
infection with C. albicans. The fungal burden of G. mellonella hemolymph was reduced, and
the administration of fluconazole—either before or after exposing the larvae, infected with
fluconazole-resistant C. albicans, to aPDT—significantly prolonged their survival compared
to the control group. These findings suggested that aPDT combined with conventional
antimicrobial drugs could have a synergistic effect, representing an effective strategy for
the treatment of infections caused by resistant clinical strains [213].
The G. mellonella model has been used to identify the regulation of innate immunity
by aPDT [93,210,216,223]. Dos Santos et al. (2017) reported that aPDT activated the G. mel-
lonella immune system by increasing the circulation of hemocytes against Porphyromonas
gingivalis infection and by attenuating infection, prolonging the survival of the infected
group of larvae [
216
]. A study by Huang et al. (2020) [
223
] confirmed that aPDT had
immunomodulatory effects; they demonstrated that 5-aminolevulinic acid (ALA)-mediated
aPDT increased hemocyte density. Moreover, the extracted hemocytes after ALA-mediated
aPDT had increased susceptibility to C. albicans and S. aureus.
Paziani et al. (2019) found that the total hemocyte count after aPDT with phenoth-
iazinium PSs (methylene blue, new methylene blue, and pentacyclic phenothiazinium
photosensitizer S137) of infected G. mellonella increased in larvae hemolymph, whereas
the fungal burden was decreased. The increase in the cellular immune response was
correlated to the increase in larval survival and decrease in fungal burden. The survival
levels of infected larvae with Fusarium keratoplasticum were 70, 60, and 80% after aPDT with
methylene blue (1500
µ
M), new methylene blue (200
µ
M), and S137 (200
µ
M), respectively,
10 days after infection. The survival levels of larvae infected with Fusarium moniliforme
were 40, 10, and 100% after aPDT with methylene blue (1500
µ
M), new methylene blue
(200
µ
M), and S137 (200
µ
M), respectively, 10 days after infection. Thus, the larvae infected
Microorganisms 2023,11, 1455 10 of 21
with F. keratoplasticum and F. moniliforme, which were found to be resistant to itraconazole and
posaconazole, survived because the cellular immune system response of G. mellonella acted
effectively [210].
Table 1summarizes a list of published works studying the effectiveness of aPDT or PS
toxicity on G. mellonella using various conditions of aPDT, tested PSs, and microorganisms
selected for infection.
Table 1.
List of published works focused on testing PS toxicity or aPDT on microbial infection using
G. mellonella larvae as a model.
Photosensitizer Light Source Energy Microorganism Authors Reference
Methylene blue
0.2 mg/mL
660 nm red light device
composed of 48 LEDs 30 J/cm2Acinetobacter baumannii Figueiredo-Godoi
et al. (2022) [18]
Fotenticine
1.2 mg/mL
660 nm red light device
composed of 48 LEDs 30 J/cm2Acinetobacter baumannii Figueiredo-Godoi
et al. (2022) [18]
Methylene blue
75–600 µM660 nm red laser light 6 J/cm2and
15 J/cm2C. albicans Figueiredo-Godoi
et al. (2019) [58]
Methylene blue
1 mM
660 ±15 nm broadband
non-coherent red light
source
0.45–18 J/cm2Enterococcus faecium Chibebe Junior
et al. (2013) [93]
Erythrosine
100 µM
532 nm green diode laser
10 J/cm2C. albicans Merigo et al. (2017) [209]
Curcumin
100 µM
405 nm blue-violet diode
laser 10 J/cm2C. albicans Merigo et al. (2017) [209]
Toluidine blue
10 µM650 nm red diode laser 10 J/cm2C. albicans Merigo et al. (2017) [209]
Methylene blue
750–3000 µM
An array of
96 light-emitting diodes
with an emission peak at
635 nm and integrated
irradiance from 570 to
670 nm
15 J/cm2Fusarium keratoplasticum,
F. moniliforme
Paziani et al. (2019)
[210]
New methylene
blue N
100–400 µM
An array of
96 light-emitting diodes
with an emission peak at
635 nm and integrated
irradiance from 570 to
670 nm
15 J/cm2Fusarium keratoplasticum,
F. moniliforme
Paziani et al. (2019)
[210]
Pentacyclic
phenothiazinium
photosensitizer S137
100–400 µM
An array of
96 light-emitting diodes
with an emission peak at
635 nm and integrated
irradiance from 570 to
670 nm
15 J/cm2Fusarium keratoplasticum,
F. moniliforme
Paziani et al. (2019)
[210]
Curcumin
50 µg/mL 440–480 nm LED source 1.2 J/cm2Streptococcus mutants Sanches et al.
(2019) [211]
Diacetylcurcumin
50 µg/mL 440–480 nm LED source 1.2 J/cm2Streptococcus mutants Sanches et al.
(2019) [211]
Methylene blue
100 µM660 nm LED source 3–18 J/cm2Escherichia coli Garcez et al. (2020) [212]
Methylene blue
1 mM
660 ±15 nm broadband
non-coherent red light
source
0.45–18 J/cm2C. albicans Chibebe Junior
et al. (2013) [213]
Microorganisms 2023,11, 1455 11 of 21
Table 1. Cont.
Photosensitizer Light Source Energy Microorganism Authors Reference
Methylene blue
600 mM 660 nm red laser light 15 J/cm2Porphyromonas gingivalis Dos Santos et al.
(2017) [216]
Curcuma longa L.
Extract
100 mg/mL
— — —Marques Meccatti
et al. (2022) [217]
Curcumin
200 µg/mL — — —Marques Meccatti
et al. (2022) [217]
Methylene blue
Concentration not
specified
An array of
96 light-emitting diodes
with an emission peak at
635 nm
15 J/cm2C. albicans, C. auris Grizante Barião
et al. (2022) [222]
New methylene
blue N
Concentration not
specified
An array of
96 light-emitting diodes
with an emission peak at
635 nm
15 J/cm2C. albicans, C. auris Grizante Barião
et al. (2022) [222]
Toluidine blue O
Concentration not
specified
An array of
96 light-emitting diodes
with an emission peak at
635 nm
15 J/cm2C. albicans, C. auris Grizante Barião
et al. (2022) [222]
Pentacyclic
phenothiazinium
photosensitizer S137
Concentration not
specified
An array of
96 light-emitting diodes
with an emission peak at
635 nm
15 J/cm2C. albicans, C. auris Grizante Barião
et al. (2022) [222]
Methylene blue
10–500 mM
630 nm red
light-emitting diode
device
Not specified Fonsecaea monophora Huang et al. (2020) [223]
5-aminolevulinic acid
10–500 mM
630 nm red
light-emitting diode
device
Not specified Fonsecaea monophora Huang et al. (2020) [223]
4. Conclusions
The information summarized in this review points to the versatile use of G. mellonella
in biological research. This model has also been proven to be highly suitable for the study
of aPDT, despite some limitations, for example, the availability of oxygen in the tissues or
the delivery of light into the tissue, while achieving high efficiency in terms of irradiation.
Of course, the biocompatibility and photoactivity of the PS are the necessary conditions
for the overall effectiveness of aPDT. Many available and generally known techniques can
be adopted with G. mellonella in terms of the experiment design and expected results, but
the protocols must be optimized, taking into consideration the specificity of this model
organism. It is also necessary to think about the fact that the G. mellonella larvae must meet
the basic standard conditions for breeding and preservation to avoid discrepancies in the
obtained results. In summary, G. mellonella has great potential for experimental studies
of aPDT.
Author Contributions:
Conceptualization, H.B., L.B. and S.K.; writing—original draft preparation,
L.B. and S.K.; writing—review and editing, H.B., L.B. and S.K. All authors have read and agreed to
the published version of the manuscript.
Funding:
This review was supported by the EU’s Next Generation EU package through the Recovery
and Resilience Plan for Slovakia under project no. 09I03-03-V01-00105 from the Government Office of
the Slovak Republic, EU grant no. 952398—CEMBO, Call: H2020-WIDESPREAD-05-2020—Twinning,
Microorganisms 2023,11, 1455 12 of 21
the Slovak Research and Development Agency under contract APVV-21-0302, grant VEGA 2/0036/22
from the Ministry of Education, Science, Research, and Sport of the Slovak Republic.
Conflicts of Interest: The authors declare no conflict of interest.
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