## 4 Discussion  
_B. xylophilus_ is a serious pest of pine trees causing PWD, which has been considered as the most destructive forest disease in China in recent years. Due to the significant economic losses caused by the PWD, many in-depth studies are being initiated to understand the disease cycle. The genetic basis is essential to study the adaptations of dispersal PWN to its host, and the interspecific communication between propagative PWN and its associated partners, and the potential role of this communication in promoting pathogenicity and  
Figure 6: Relative expression levels of \(HSP90\) under different temperature treatments. The relative mRNA expression levels of \(HSP90\) were normalized according to the most suitable ((**a**), \(UBCE\) and \(EF1\gamma\)) and the least suitable ((**b**), \(UBQ\) and \(18S\)\(rRNA\)) reference genes, respectively. Bars show the means and SEs. Different letters indicate significant differences (\(p<0.05\), one-way analysis of variance followed by LSD Multiple Comparison). Figure 7: Relative expression levels of \(AK\) in different developmental stages. The relative mRNA expression levels of \(AK\) were normalized according to the most suitable ((**a**), \(EF1\gamma\) and \(Actin\)) and the least suitable ((**b**), \(UBQ\) and \(18S\)\(rRNA\)) reference genes, respectively. Bars show the means and SEs. Different letters indicate significant differences (\(p<0.05\), one-way analysis of variance followed by LSD Multiple Comparison). invasiveness of PWN. Most of these studies involve qRT-PCR technique to quantify the expression of target genes efficiently and accurately. This technique relies on the stable expression of reference genes to eliminate the impact of differences in RNA quality, reverse transcription efficiency, and human factors. Hence, the selection of the most appropriate and stable reference genes is the basis of qRT-PCR success [44]. Because the expression of reference genes of interest is influenced by experimental conditions [45; 46; 47], the screening of reference genes is particularly necessary for quantitative genetic studies. Based on the transcriptome data of pine wood nematodes, we have screened candidate reference genes of _B. xylophilus_ suitable for studying the effects of temperature variation and developmental stages. The suitable reference genes vary according to species, developmental stages, and temperature used, indicating that there is no absolute universality between the homologous reference genes of different insect species [48; 49; 50; 51]. In order to overcome this limitation, some studies have screened internal reference genes of insects in different conditions, such as selecting reference genes in different tissues and geographical populations of _Nilaparata lugens_[52] or screening reference genes in _Helicoverpa armigera_ at different temperatures [53]. Our current study is the first one to evaluate different internal reference genes of _B. xylophilus_ in varying developmental stages and temperatures, and to report their relative stability. The stability of the eight reference genes selected varied based on the chosen analysis method (Table 2). It is difficult to identify reference genes which are generally stable across these four algorithms, as each program has its own strengths and appropriate application conditions [29; 54; 55]. When compared across different temperature treatments, the results of geNorm were distinct from those of delta Ct, BestKeeper, and NormFinder (Table 2). Nonetheless, there was also partial consistency. As shown in Table 2, the stable combination of reference genes for temperature treatments was _UBCE_ and _EF1\(\gamma\)_, whereas _UBQ_ and _18S rRNA_ were the most unstable reference genes based on all four statistical algorithms. In the analysis of the developmental stages, the \(\Delta\)Ct method and NormFinder represented that _EF1\(\gamma\)_ and _UBCE_ were the most stable genes. Based on BestKeeper, _PMP-2_ and _Actin_ were the appropriate reference genes (Table 2).