Phagocytic activity of haemocytes from Galleria mellonella larvae infected with Candida glabrata, Candida nivariensis and Candida bracarensis
Ainara Hernando, Estibaliz Mateo, Elena Eraso and Guillermo Quindós
Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y Enfermería, Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), Bilbao, Spain (GIC15/78 IT-990-16, UFI 11/25)
Objectives: To assess the phagocytic capacity of haemocytes from Galleria mellonella larvae infected with Candida glabrata, Candida nivariensis and Candida bracarensis. 
Methods: Galleria mellonella larvae were distributed in groups of five and infected with 10 µl of 1x106 cells/ml of each of the following reference strains: Candida glabrata ATCC 90030, Candida glabrata NCPF 3203, Candida nivariensis CBS 9984, Candida nivariensis CECT 11998, Candida bracarensis NCYC 3397 and Candida bracarensis NCYC 3133. Yeast cells were previously stained with Calcofluor White for 30 min at 37 ºC and washed twice in PBS buffer. Then, larvae were injected with Candida in the last left pro-leg using a precision syringe and incubated for 2 h at 37 ºC. Afterwards, haemolymph of each larva was collected and 50 µl were diluted in the same volume of IPS buffer (Insect Physiological Saline buffer) to avoid melanisation. The phagocytic activity of the haemocytes was determined using a fluorescence microscope. Phagocytosis quantification was performed counting the percentage of haemocytes with cells phagocytised and without cells phagocytised. Experiments were performed at least three times on different days and, at least, 100 haemocytes were counted. The results obtained were analysed using one-way ANOVA to evaluate the differences between strains (p < 0.05 was considered as statistically significant).
Results: Larvae infected with both Candida glabrata strains presented the same phagocytosis capacity and no statistically significant differences in the phagocytosis percentage between them were observed (Table 1). The same results were obtained in larvae infected with Candida bracarensis strains, without differences between them. However, the phagocytosis obtained with Candida bracarensis NCYC 3397 showed differences with those observed in larvae infected with Candida glabrata and Candida nivariensis. Regarding to Candida nivariensis, differences were observed in the phagocytosis percentage between these two strains. Moreover, results obtained after infection with Candida nivariensis CECT 11998 presented differences in phagocytosis percentage with respect to all analysed Candida strains. 
Conclusions: The phagocytic activity of haemocytes from infected Galleria mellonella larvae is a useful tool to study invasive candidiasis caused by Candida glabrata, Candida nivariensis and Candida bracarensis. The phagocytosis activities observed in Galleria mellonella larvae infected with Candida were similar for all strains tested except for Candida bracarensis NCYC 3397 and for Candida nivariensis CECT 11998. 
Funding: This study was partially financed by GIC15/78 IT-990-16 (Gobierno Vasco-Eusko Jaurlaritza). AH has a grant from the UPV/EHU.
 
Table 1: Phagocytic activities of haemocytes from larvae infected with 1x106 cells/ml of Candida glabrata, Candida nivariensis and Candida bracarensis. 
Candida strains	Phagocytosis (%)
Candida glabrata ATCC 90030	8.02*/**
Candida glabrata NCPF 3203	7.91*/**
Candida nivariensis CBS 9984	7.82*/**
Candida nivariensis CECT 11998	4.32**
Candida bracarensis NCYC 3397	10.20*
Candida bracarensis NCYC 3133	8.33*
* statistically significant differences with Candida nivariensis CECT 11998
** statistically significant differences detected with Candida bracarensis NCYC 3397