In this study we used the invertebrate model G. mellonella for the in vivo study of antifungal PDT. We verified that aPDT prolonged the survival of G. mellonella caterpillars infected by C. albicans and reduced the fungal burden in the hemolymph of these animals. In addition, we used a fluconazole-resistant C. albicans strain to test the combination of aPDT and fluconazole. The data presented here demonstrated that aPDT increased the susceptibility of C. albicans to fluconazole.
The increased numbers of fungal infections and the subsequent need for high-cost and time-consuming development of new antimicrobial strategies and anti-infectives has emerged as a major problem among infectious diseases researchers and clinicians
[6, 26]. Antimicrobial PDT is one of the most promising alternative countermeasures for cutaneous or mucosal infections, caused by either bacteria or fungi
Antifungal PDT is an area of increasing interest, as research is advancing in answering fundamental questions regarding the photochemical and photophysical mechanisms involved in photoinactivation; producing new, potent and clinically compatible PS; and in understanding the effect of key microbial phenotypic multidrug resistance, virulence and pathogenesis determinants in photoinactivation. The novel concept of developing the non-vertebrate infection model in G. mellonella to explore the efficacy of antifungal PDT provides many competitive advantages
The use of the invertebrate model host has significant benefits when compared to mammalian animals: there are no ethical or legal concerns, no need for specialized feeding or housing facilities, the management of the animal is very easy and no anesthesia is needed, animals are inexpensive, and the use of large sample numbers in the same group are possible
[27–30]. G. mellonella has been used to study host-pathogen interactions as an alternative host model to small mammals such as mice and rats
[9, 27–29, 31–40].
Our laboratory pioneered the use of G. mellonella as a suitable invertebrate model host to study aPDT against Enterococcus faecium. In the present study this approach to investigating aPDT was successfully expanded to include fungal pathogens. The optimal dose–response to MB mediated-PDT was evaluated and 0.9 J/cm2 showed the best survival of G. mellonella caterpillars, as was found in the E. faecium study. The same limited non-toxic dosage of aPDT to G. mellonella was applied to treat larvae infected by strains of Candida albicans.
During the G. mellonella killing assays, groups infected by C. albicans that received aPDT treatment demonstrated prolonged survival when compared to groups that did not received treatment. However a statistically significant difference between PDT and control groups was observed only for C. albicans Can14 wild-type strain. When the infection was induced by a fluconazole resistant strain (Can37), a statistically significant difference between these groups was not observed. Despite the fact that PDT has been described as a potent agent against both antimicrobial-resistant and sensitive microorganisms
 we observed that a fluconazole-resistant C. albicans strain was less sensitive to aPDT.
This difference has also been described in an in vitro study performed by Dovigo et al.
. These authors observed that fluconazole-resistant strains of C. albicans and C. glabrata showed reduced sensitivity to aPDT in comparison with reference strains susceptible to fluconazole, suggesting that resistance mechanisms of microorganisms to traditional antifungal drugs could reduce PDT effectiveness. According to Prates et al.
, the resistance of Candida strains to fluconazole usually involves overexpression of cell membrane multidrug efflux systems belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily (MFS) classes of transporters. The authors showed that the overexpression of both systems reduced MB uptake by fungal cells, as well as the killing effect of aPDT, suggesting that ABCs and MFSs are involved in the efficiency of aPDT mediated by MB and red light. In addition, Arana et al.
 demonstrated that subinhibitory concentrations of fluconazole induced oxidative stress and a transcriptional adaptative response that was able to generate protection of C. albicans against subsequent challenges with oxidants. The mechanisms of protection against oxidative stress of fluconazole resistant C. albicans strain may have enhanced the resistance of C. albicans to oxidative damage caused by PDT.
In this study, we also evaluated the effects of aPDT on fungal cells in the hemolymph of G. mellonella larvae infected by fluconazole resistant C. albicans (Can37). Although this C. albicans strain had not shown a significant increase in survival rate in G. mellonella, it was observed that aPDT caused a reduction of the number of fungal cells in the hemolymph (0.2 Log) with a statistically significant difference between aPDT and control groups. In addition, these data demonstrated that aPDT was able to reduce fungal cell viability immediately upon light exposure, suggesting that C. albicans cells were sensitive to aPDT, by the lethal oxidative damage of the singlet oxygen pathway, in the experimental candidiasis in the G. mellonella model. At the moment, all the aPDT studies performed in vivo were developed in vertebrate models of rats and mice using fluences of light much higher than the dose used in our work
[43–45]. Using an oral candidiasis mice model, Costa and colleagues
 found a reduction of 0.73 Log in the fungal cells recovered after erythrosine- and LED-mediated aPDT when a fluence of 14 J/cm2 was applied. Dai et al.
 also demonstrated that aPDT, with the combination of methylene blue and red light (78 J/cm2), reduced (0.77 Log of CFU) the fungal burden in skin abrasion wounds in mice infected with C. albicans.
Patients with fungal infections are often treated with azole antifungal drugs, however Candida resistance to azoles has been detected in recent years. Several mechanisms of resistance have been reported including the overexpression of cell membrane multidrug efflux pumps previously cited, an alteration in the chemical structure of the demethylase enzyme, and the incorporation of alternative sterols to ergosterol within the cell membrane
[23, 24]. Giroldo et al.
 suggested that MB-mediated aPDT caused damage to the cell membrane of the C. albicans cells. If the hypothesis that aPDT could affect the cell membrane is valid, the sequential use of aPDT with fluconazole could have a dual action on treating the infection. Conventional antimicrobial therapy could have aPDT as an adjunct or as an alternative
. The combination of PDT with antimicrobials has been used with success when compared to either isolated approach
[19, 26, 46]. Kato et al.
 verified that after exposure to sublethal aPDT, the minimal inhibitory concentration (MIC) of fluconazole against C. albicans was reduced compared to non-aPDT treated strains.
Of note, we observed that the G. mellonella larvae survival after infection by the fluconazole resistant C. albicans strain, was prolonged when fluconazole was administered before or after aPDT, in comparison to the use of fluconazole or PDT alone. We believe that due to the permeabilization of the fungal cell membrane by the sublethal PDT dose, fungal cells become more susceptible to fluconazole action. In addition, it has been suggested that the use of azoles can increase the oxidative stress promoted by PDT by contributing to ROS formation themselves
. Arana et al.
 demonstrated that fluconazole was able to induce oxidative stress in C. albicans in a dose- and time-dependent manner, suggesting that ROS play a role in the mechanism of action of azoles. The exact mechanism involved in increasing the survival of larvae infected by the fluconazole resistant C. albicans strain and exposed to combined therapy of PDT and fluconazole remains to be clarified. Thus, comprehensive experiments are needed to better understand whether this process could be useful to treat antimicrobial resistant fungal infections.
In summary, the results obtained in this study showed that G. mellonella is a suitable model host to study the antifungal PDT in vivo. It is known that the G. mellonella model is not restricted to studies that examine aspects of the pathogenesis of fungal infections or antimicrobial therapies, but also can be used to the study of host defenses against fungal pathogens . The insect immune response demonstrates a number of strong structural and functional similarities to the innate immune response of mammals and, in particular, insect haemocytes and mammalian neutrophils have been shown to phagocytose and kill pathogens in a similar manner . Recent studies demonstrated that PDT can stimulate host defense mechanisms. Tanaka et al.  used a murine methicilin-resistant Staphylococcus aureus (MRSA) arthritis model and verified that the MB-mediated PDT exerted a therapeutic effect against a bacterial infection via the attraction and accumulation of neutrophils into the infected region. Neutrophils are among the first cells recruited to the illuminated area and their main function is to release enzymes for killing infectious organisms and secrete cytokines and other chemicals that promote inflammation . In this study, the effects of aPDT on the immune system of G. mellonella were not investigated. Therefore, future studies need to be developed to understanding the action of aPDT and methylene blue in the haemocyte density and in the expression of a variety of antimicrobial peptides involved in immune responses of G. mellonella.
The key conclusion is that the G. mellonela - C. albicans system is a suitable model to study antifungal PDT and to explore combinatorial aPDT-based treatments. Thus, this invertebrate animal model host provides a novel approach to assess the effects of in vivo PDT, alone or in combination with antifungal compounds, on fungal infections without the difficulties of mammalian models.