Fungal G-protein coupled receptors (GPCRs) play essential roles in sensing environmental cues including host signals.The study of GPCR in mediating fungus-insect interactions is still limited.Here we report the evolution of GPCR genes encoded in the entomopathogenic Metarhizium species and found the expansion of Pth11-like GPCRs in the generalist species with a wide host range.By deletion of ten candidate genes MrGprl-MrGpr10 selected from the six obtained subfamilies in the generalist M.robertsii,we found that each of them played a varied level of roles in mediating appressorium formation.In particular,deletion of MrGpr8 resulted in the failure of appressorium formation on different substrates and the loss of virulence during topical infection of insects but not during injection assays when compared with the wild-type (WT) strain.Further analysis revealed that disruption of MrGpr8 substantially impaired the nucleus translocation of the mitogen-activated protein kinase (MAPK) Mero-Fus3 but not the MAPK Mero-Slt2 during appressorium formation.We also found that the defect of △MrGpr8 could not be rescued with the addition of cyclic AMP for appressorium formation.Relative to the WT,differential expression of the selected genes have also been detected in △MrGpr8.The results of this study may benefit the understanding of fungus-interactions mediated by GPCRs.