Strains of microorganism
A clinical isolate of F. monophora was recovered at Sun Yat-sen Memorial Hospital, Guangzhou, China, and the identification was carried out as described previously [8]. In order to induce the formation of conidia, the F. monophora isolate was subcultured from Sabouraud dextrose agar to potato dextrose agar (Sigma-Aldrich, St. Louis, MO, USA), and kept at 26 °C for 10 days. Standard strains of C. albicans (ATCC 18804) and S. aureus (ATCC 12600) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and stored in frozen stocks with BHI broth (Sigma-Aldrich, St. Louis, MO, USA), containing 20% glycerol at − 80 °C. S. aureus was grown on LB plates (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 24 h before the use.
G. mellonella infection models and the killing assay
The G. mellonella infection was carried out as described previously [12]. Briefly, G. mellonella in the final instar larval stage (300–350 mg in body weight) were selected and distributed randomly into different groups. Every infection set consisted of 5 larvae. Triplicate sets of 15 larvae were employed for every condition in all killing assays. Ten μL of the conidia inoculum with 1 × 106 cells were injected into the hemocoel of the larvae via the last left proleg. The same volume of PBS (Thermo Scientific, Wilmington, DE, USA) was injected as the control. After inoculation, the infected larvae were placed in disposable Petri dishes and incubated at 37 °C. The number of dead larvae was scored daily for the survival curve. The larvae were considered dead when they displayed no movement in response to touch and removed from the dishes.
Application of photodynamic therapy on G. mellonella
The fresh solution of photosensitizers MB and ALA (Sigma-Aldrich, St. Louis, MO, USA) were prepared for each experiment. A volume of 10 μL of the photosensitizer solution at different concentrations (from 10 mM to 500 mM) was injected into the larvae via the last right proleg. Every treatment set consisted of five larvae. Triplicate sets of 15 larvae were performed. Then the larvae were maintained in the dark for 30 min as the pre-irradiation time. The laser light (PDT, LED-IB, Wuhan Yage, China) was applied to larvae contained in the 6-well plate for 25 min. After irradiation, larvae were transferred to the dark incubator and kept at 37 °C for the required time. For larvae infected with F. monophora, the application of PDT on larvae was performed 2 h after inoculation with fungal spores.
Quantification of G. mellonella hemocytes
A volume of 10 μL of photosensitizer ALA at the concentration of 100 mM was used for PDT treatment on G. mellonella. The larvae were exposed to red light 30 min after the injection of ALA. Then, the treated larvae were incubated at 37 °C for 4 h, and hemolymph was extracted. Every treatment set consisted of six larvae and repeated trice. The hemolymph was collected into 1.5 mL Eppendorf Safe-Lock Microcentrifuge Tube (Sigma-Aldrich, St. Louis, MO, USA) containing ice-cold Grace’s medium (Thermo Scientific, Wilmington, DE, USA). Then hemocytes were purified after centrifugation and quantified by the hemocytometer.
The disk diffusion assay
Isolate of C. albicans and S. aureus was respectively grown in YPD and LB liquid medium (Sigma-Aldrich, St. Louis, MO, USA) overnight. The next day about 1 × 105 yeast and bacterial cells were spread on a rich medium plate (10 cm Petri dish), separately [21]. Hemolymph was extracted from larvae 4 h post-PDT and diluted with PBS in the ratio of 1:1. Every treatment set consisted of five larvae. Triplicate sets of 15 larvae were performed. The blank round paper disks (eight mm in diameter) were impregnated with prepared hemolymph or PBS control. Then, prepared disks were placed triangularly on C. albicans and S. aureus plates, and the plates were incubated in the dark at 35 °C for 24 h. Then photographed the plates and compared the diameters of the inhibition zones. Each assay was performed thrice.
The in vitro bacterial and fungal killing assay
Fifteen larvae per group with or without PDT treatment were used for colony-forming units (CFU) counting. The hemolymph of larvae was collected 4 h post light exposure for killing assay. Hemocytes (1 × 107) were incubated with S. aureus (2 × 107) or C. albicans (2 × 107) in a chamber at 37 °C. The killing of S. aureus and C. albicans were measured every 15 min. The survival observation was performed through CFU determination. The survival rate was calculated by CFU at every time point comparing with the initial bacterial load.
Microscopy
Hemolymph was extracted from G. mellonella 4 h post-ALA-PDT and collected for purifying hemocytes. Bacterial cells of S. aureus were stained with Wheat Germ Agglutinin (WGA) labeled with Biotium’s Dye CF555 (Biotium, Inc., Hayward, CA, USA) at 37 °C in the dark for 15 min, and co-cultured with hemocytes at the ratio of 2:1 in the 96-well plate for 3 h. After washing with PBS thrice, hemocytes were stained with DAPI (Thermo Scientific, Wilmington, DE, USA) in the dark for 10 min, then mounted on glass coverslips and viewed directly using an Olympus BX63 fluorescence microscope.
Statistical analysis
Data were analyzed by using the SPSS software 15.0 (SPSS Inc., Chicago, IL, USA). G. mellonella survival was examined using the Kaplan-Meier method, and differences were determined by using the log-rank (Mantel Cox) test. Difference in hemocytes density and bacterial/fungal killing assays was assessed by ANOVA analysis followed by the Tukey’s test. A p-value of 0.05 in all replicate experiments was considered statistically significant.