Haemocyte responses in Galleria mellonella infected by 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)
Objective: To determine the haemocyte responses during invasive infections caused by Candida glabrata, Candida nivariensis and Candida bracarensis in a Galleria mellonella model.
Methods: Galleria mellonella larvae were distributed in groups of five and infected with 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. Larvae were inoculated with 10 µl of 1x105, 1x106 and 1x107 yeast/ml in the last left pro-leg with a precision syringe. Two control groups were used: untouched larvae and larvae inoculated with PBS buffer supplemented whit ampicillin (20 µg/ml). After infection, larvae were incubated for 3 h at 37 ºC and then, haemolymph of each larva was collected. The same volume of haemolymph, 50 µl, was mixed with IPS buffer (Insect Physiological Saline buffer) to avoid melanisation. The haemocyte response was determined by counting cells using a haemocytometer. Each assay was repeated at least three times. The results obtained with each inoculum 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 behaviour: statistically significant differences in the response of haemocytes were observed between the untouched larvae and those infected with the 1x105 and 1x106 inocula, but not differences were detected with 1x107 cells/ml inoculum (Table 1). Similar results were obtained in larvae infected with Candida nivariensis CECT 11998 but not with CBS 9984 strain, in which differences were detected with 1x107 and 1x105 cells/ml but not with 1x106 cells/ml. Otherwise, haemocyte responses of larvae infected with Candida bracarensis strains presented differences with untouched larvae; however, no significant differences between larvae infected with all the assayed inocula were showed.
Conclusions: Galleria mellonella is a useful model to study invasive candidiasis throughout analysis of haemocyte responses to the infections caused by Candida glabrata, Candida nivariensis and Candida bracarensis. The haemocytes responses in Galleria mellonella larvae infected with Candida glabrata and Candida nivariensis was similar but differences between the inocula injected were observed. However, there were no differences in the haemocytes production in larvae infected with Candida bracarensis. 
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: Haemocyte responses in Galleria mellonella larvae infected with 1x105, 1x106 and 1x107 cells of Candida. Statistically significant differences between untouched larvae and larvae infected with Candida were annotated with asterisk.
Candida strains	Inocula
(Candida cells/ml)	Haemocytes/ml
Control 1
Untouched larvae	8.66x106
Control 2
PBS+ampicillin (20 µg/ml)	1.77x107
Candida glabrata 
ATCC 90030	1x105	2.58x107*
	1x106	2.23x107*
	1x107	1.31x107
Candida glabrata 
NCPF 3203	1x105	2.47x107*
	1x106	2.15x107*
	1x107	1.21x107
Candida nivariensis 
CBS 9984	1x105	2.08x107*
	1x106	1.38x107
	1x107	1.68x107*
Candida nivariensis 
CECT 11998	1x105	2.39x107*
	1x106	1.50x107*
	1x107	1.17x107
Candida bracarensis 
NCYC 3397	1x105	2.26x107*
	1x106	2.06x107*
	1x107	2.15x107*
Candida bracarensis
NCYC 3133	1x105	2.14x107*
	1x106	2.03x107*
	1x107	2.05x107*