Background: The pine wood nematodeBursaphelenchus xylophilusis a destructive pest ofPinustrees worldwide and lacks effective control measures. Screening for nematotoxic proteins has been undertaken to develop new strategies for nematode control.Results: The results of the present study provided initial insights into the responses ofB. xylophilusexposed to a nematotoxic cytolytic-like protein (CytCo) based on transcriptome profiling. A large set of differentially expressed genes (DEGs = 1265) was found to be related to nematode development, reproduction, metabolism, motion, and immune system. In response to the toxic protein,B. xylophilusupregulated DEGs encoding cuticle collagens, transporters, and cytochrome P450. In addition, many DEGs related to cell death, lipid metabolism, major sperm proteins, proteinases/peptidases, phosphatases, kinases, virulence factors, and transthyretin-like proteins were downregulated. Gene Ontology enrichment analysis showed that the CytCo treatment substantially affected DEGs involved in muscle contraction, lipid localization, and the mitogen-activated protein kinase cascade. The pathway richness of the Kyoto Encyclopedia of Genes and Genomes showed that the DEGs were concentrated in lysosomes and involved in fatty acid degradation. Weighted co-expression network analysis indicated that the hub genes affected by CytCo were associated with the nematode cuticular collagen.Conclusions: These results showed that CytCo toxin interferes with gene expression to exert multiple nematotoxic effects, thereby providing insights into its potential use in pine wood nematode control.

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5′-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 µg/mL) in the soaking buffer for 24 h in the dark at 25 °C. The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.