Debilitation of Galleria mellonella hemocytes using CytCo a cytolytic-like protein derived from the entomopathogen Conidiobolus obscurus.

Cytolytic (Cyt)-like genes are present in both pathogenic bacteria and fungi. Bacterial Cyt proteins can destroy insect midgut epithelial cells after ingestion by hosts and some of them have been developed as biopesticides; however, few studies have investigated their functions in fungal pathogens. This study investigated the effects of a Cyt-like protein (CytCo) derived from Conidiobolus obscurus (Entomophthoromycotina) on the hemocytes of the greater wax moth Galleria mellonella larvae. The results showed a significant decline in hemocyte viability after treatment with CytCo in vivo or in vitro. The hemocyte density in the hemolymph was reduced by 65.2% and 50.2% after 12 h in vivo and 6 h in vitro treatments, respectively. Apoptosis/necrosis tests using fluorescence microscopy demonstrated that CytCo-treated hemocytes displayed apoptosis, and many of them also showed necrosis after 6 h in vitro treatment. Based on transcriptome analysis, several genes involved in the programmed cell death signaling pathway were upregulated in the CytCo-treated hemocytes. Meanwhile, the differentially expressed genes related to energy production, signal transduction, transcription regulation, and melanization were upregulated, demonstrating activated immune responses; those putatively related to hemocyte adhesion were downregulated, possibly in response to the reduction of hemocytes in hemolymph. In conclusion, CytCo as a virulence factor, could irreversibly incapacitate host hemocytes, playing an important role in debilitating insect immunity. This novel insecticidal protein holds a potential to develop biopesticide for controlling agroforestry pests.