Selection and Validation of Reliable Reference Genes for qRT-PCR Normalization of Bursaphelenchus xylophilus from Different Temperature Conditions and Developmental Stages

Round 1

Reviewer 1 Report

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Reviewer 2 Report

Dear authors
Would you be willing to add to the discussion on the sensitivity and specificity of molecular methods currently used to detect Bursaphelenchus xylophilus? If confounding (false positive identifications) occurs, e.g., due to the presence of other related nematode species, there are consequences resulting from quarantine regulations and, e.g., timber export embargoes. In Europe, despite million-euros efforts to stop the spread of PWN in Portugal (felling trees and shrubs in strips several kilometres long and restricting the Monochamus population), nematode transmission to Spain occurred. Therefore, it is important to highlight the potential practical aspect of your research. The possibility of early detection of different developmental forms of nematodes in environmental samples and considering temperature conditions is crucial in this case.
If the reference genes studied vary according to species, developmental stage, and temperature, suggesting that there is no absolute universality between homologous reference genes of different insect species, how can this problem be overcome in practise? In addition, the stability of the eight selected reference genes differed depending on the analytical method chosen (Table 2). In your opinion, does this fact represent a major problem? From a practical point of view, should one or more methods be chosen? Should both quarantine services and laboratories of forest science organisations have standard methods so that they are comparable? Who should provide a comprehensive ranking based on the results of different methods to provide a reliable indication of target gene stability? Will this be the focus of your further research on molecular mechanisms across functional and developmental genomics of nematodes.
Can you describe the potential relevance of your research findings to practical applications, e.g., in forestry, the importance of identifying appropriate and reliable reference genes? How will the development of a standardised RT -qPCR analysis for the pine nematode contribute to the control of this pest?

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Reviewer 3 Report

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