Establishment and characterization of a Monochamus alternatus cell line for molecular biological analyses

Pine wilt disease (PWD) is one of the most destructive global forest diseases, causing extensive mortality in pine trees and severe economic and ecological damage. PWD is caused by the pinewood nematode Bursaphelenchus xylophilus, which is transmitted by Monochamus beetles. Despite the critical role of M. alternatus in PWD epidemiology, molecular and cellular studies of this species have been severely limited by the lack of a continuous cell line suitable for functional analyses. In this study, the first continuous insect cell line derived from M. alternatus, designated NARO-Moal, was produced from prepupal fat bodies. The cell line has been stably maintained for over 50 passages and adapted to the commercially available Grace's Insect Medium. To provide a molecular framework for functional studies, de novo transcriptome sequencing of NARO-Moal cells generated an assembly of 14,141 genes with 89.5% completeness, based on BUSCO analysis. The NARO-Moal cells supported transient gene expression following plasmid transfection and showed efficient RNA interference in response to simple soaking with double-stranded RNA. Moreover, this cell line responded to juvenile hormone at the gene expression level and supported stable infection with Wolbachia. These results revealed that the NARO-Moal cell line is a versatile molecular and cellular platform for gene functional analysis, endocrine signaling, and symbiotic research in M. alternatus, providing new opportunities for mechanistic studies and the development of innovative strategies to control pine wilt disease.