###### Abstract  
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a powerful technique for studying gene expression. The key to quantitative accuracy depends on the stability of the reference genes used for data normalization under different experimental conditions. Pine wood nematode (PWN, _Bursaphelenchus xylophilus_) is the causal agent of the devastating pine wilt disease (PWD). Extensive and prompt research is needed to understand the molecular mechanism of PWD, but identification of the reference PWN genes for standardized qRT-PCR has not been reported yet. We have analyzed eight candidate reference genes of PWN across different temperature conditions and developmental stages. Delta Ct method, GeNorm, NormFinder, BestKeeper, and Reffinder algorithms were used to evaluate the stability of expression of these genes. Finally, we use heat shock protein 90 (_HSP90_) in different temperatures and arginine kinase gene (_AK_) in different developmental stages to confirm the stability of these genes. _UBSE_ and _EF17_ were most stable across different temperature treatments, whereas _EF17_ and _Actin_ were most stable across different developmental stages. In general, these results indicate that _EF17_ is the most stable gene for qRT-PCR under different conditions. The systematic analysis of qRT-PCR reference gene selection will be helpful for future functional analysis and exploration of _B. xylophilus_ genetic resources. Bursaphelenchus xylophilus; reference gene; qRT-PCR; normalization +
Footnote †: journal: _Sci._  
Footnote †: journal: _Sci. with the suitable developmental stages of the vector beetles, i.e., late pupal or early adult phase, PWN holds into fourth juvenile (LIV) dispersal stage and enters the beetle's tracheal system, inside of which the nematodes are transported to new pine hosts [2,5]. Because the pathogenesis of _B. xylophilus_ and the virulence of PWD are complex and interact strongly with the vector beetle, associated fungi and prevailing environmental conditions, detailed study of _B. xylophilus_ is necessary for the development of new PWD control methods. To manage this destructive disease, extensive efforts have been made to control PWN, chemical control being one of the most common approaches. However, the indiscriminate use of pesticides has not only posed environmental hazards, but also lead to the emergence of pesticide resistance in PWN [8]. Developing environmentally friendly pest control methods, such as RNAi or nematophagous fungal formulations, are necessary alternative solutions. Considering the economic importance and the urgency of developing effective and efficient control strategies, further ecological, physiological, and molecular biological research on _B. xylophilus_ has been extensively conducted over the past few decades [9, 10, 11, 12, 13, 14]. However, further studies involving various aspects of the molecular mechanism of PWN, including development, immunity, energy metabolism, reproduction, chemoreception, and other biological process are required [15, 16, 17, 18]. To achieve these research goals, analysis of the regulation and expression patterns of functional genes is essential. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) has been widely used for gene expression analysis owing to its high specificity, rapidity, reproducibility, and low cost [19, 20, 21]. An indispensable factor for the qRT-PCR method is to select a gene with a stable expression and use it as a reference to reduce the differences between samples through analysis and calculation to ensure the confidence of the final result [22]. Common internal reference genes generally include elongation factor (_EF_), actin (_ACT_), 185 rRNA, etc. [23, 24, 25]. However, the stability of the reference gene is affected by various factors such as temperature, development stage, and tissue type.