Galleria mellonella—A Model for the Study of aPDT—Prospects and Drawbacks

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.


Introduction
In modern medicine and pharmaceutical research, the selection of the appropriate choice of in vivo model has been critical [1][2][3][4][5][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][17][18][19][20][21][22].
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 effectiveness 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 Escherichia 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 deposition. 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].
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. mellonella 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][154][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-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][152][153][154], the immune response mechanism shows signs related to the innate immunity of vertebrates [156][157][158][159]. Hemocoel contains hemocytes with a similar function to human neutrophils that participate in phagocytosis, during which reactive oxygen species (ROS) are generated [160][161][162][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].
Several approaches are currently available, and these have been adapted and optimized 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 infection/treatment; (ii) the host-pathogen interaction. The latter scope includes quantitative analysis, but also qualitative ones that consider hemolymph analysis. Both areas can also involve 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][169][170][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][176][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][179][180][181][182][183][184].
The implementation of molecular biology is necessary to obtain more detailed information 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][185][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 regulatory 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 (malonic 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 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.

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][27][28][199][200][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][205][206][207][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 investigate 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][210][211][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 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 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 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. during infections caused by bacterial and fungal-mono-but also dual or multi-species biofilms [18,58,[209][210][211][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 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 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 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. 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 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 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 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.
Microorganisms 2023, 11, x FOR PEER REVIEW 7 ( Figure 2). To prevent the organism from moving around, it is advisable to perform irradiation of each larva separately, one by one, and to keep the larva inside its well u forceps. After performing aPDT, the larvae are incubated in Petri dishes at the requ temperature (usually at 37 °C in the dark). Every experiment must include a control gr of G. mellonella larvae that do not receive any injections to monitor the overall qualit the larvae over the course of the experiment, as well as a PBS injection control grou ensure that death was not due to trauma. The survival of aPDT-treated larvae is recor daily or hourly, according to a pathological scoring system proposed by Loh et al. (2 [78] taking into account a few attributes, such as movement activity, melanization, or coon formation. Figure 2 illustrates the irradiation of G. mellonella larvae with a red la and how this is processed in the laboratory of Prof. Bujdáková et al. The key factor in PDT (aPDI) effectiveness is PS, which must meet the compatib parameters and have high efficiency. Absorption in the red and near-infrared spectru also advantageous, as red light is relatively favorable to the treated host. The PS need exhibit only local toxicity, even after light activation. A high level of ROS yield is assumed during irradiation [219][220][221].
Phenothiazinium dyes are the most common PSs used in PDT performed on th mellonella model [58,210,212,216,222]. During the administration of the desired PS into larva´s hemocoel, the body of the larva becomes colored, which is an accompany 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][220][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). 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). 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 localization of porphyrin PS was assessed using fluorescence microscopy after the hemocytes were isolated and collected from the hemolymph of inoculated larvae [226].
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 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 localization of porphyrin PS was assessed using fluorescence microscopy after the hemocytes were isolated and collected from the hemolymph of inoculated larvae [226].
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].  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 erythrosine) 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 µM) 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 2.5 mm. In association with 100 µM 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 appropriate 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  reported that aPDT activated the G. mellonella 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 phenothiazinium 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 µM), new methylene blue (200 µM), and S137 (200 µM), 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 µM), new methylene blue (200 µM), and S137 (200 µM), respectively, 10 days after infection. Thus, the larvae infected 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 1 summarizes 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.

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.