Stability of nuclear and mitochondrial reference genes in selected tissues of the ambrosia beetle Xylosandrus germanus

Authors: PATWA, NISHA - Oak Ridge Institute For Science And Education (ORISE); Ranger, Christopher; LEHENBERGER, MAXIMILIAN - Max Planck Institute Of Chemical Ecology; BIEDERMANN, PETER - University Of Freiburg; Reding, Michael - Mike

Submitted to: Insects
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2021
Publication Date: 12/16/2021
Citation: Patwa, N., Ranger, C.M., Lehenberger, M., Biedermann, P., Reding, M.E. 2021. Stability of nuclear and mitochondrial reference genes in selected tissues of the ambrosia beetle Xylosandrus germanus. Insects. 12(12). Article 1125. https://doi.org/10.3390/insects12121125.
DOI: https://doi.org/10.3390/insects12121125

Interpretive Summary: The ambrosia beetle Xylosandrus germanus is a destructive wood-boring insect of horticultural tree crops. A fungal mutualist is cultivated within host trees that provides the sole source of nutrition for the larvae and adults. Female X. germanus adults use a pouch-like structure (i.e., mycetangium) to maintain and transport spores of their fungal mutualist. To facilitate future studies examining gene expression of X. germanus’ mycetangia, the identification of stable genes unaffected by experimental treatments is needed to provide a standard reference during gene expression studies. Selected tissue types were dissected from laboratory-reared and field-collected specimens of the ambrosia beetle X. germanus to evaluate the stability of five reference genes, namely, 28S ribosomal RNA (28S rRNA), arginine kinase (AK), carbamoyl-PO4 synthase 2 (CAD), mitochondrial cytochrome oxidase 1 (CO1), and elongation factor-1a (EF1a). The reference genes CO1 and AK were identified as primary and secondary reference genes. By contrast, EF1a was considered unsuitable for use as a reference gene during gene expression studies with X. germanus. These results will aid in normalizing the expression of target genes during studies with X. germanus.

Technical Abstract: The fungus-farming ambrosia beetle Xylosandrus germanus uses a pouch-like structure (i.e., mycetangium) to transport spores of its nutritional fungal mutualist. Our current study sought to identify reference genes necessary for future transcriptome analyses aimed at characterizing gene expression within the mycetangia. Complementary DNA was synthesized using selected tissue types from laboratory-reared and field-collected X. germanus consisting of the whole body, head + thorax, scutellum + deflated or inflated mycetangia, inflated mycetangia, and abdomen. Quantitative reverse-transcription PCR (RT-qPCR) reactions were performed using primers for 28S ribosomal RNA (28S rRNA), arginine kinase (AK), carbamoyl-PO4 synthase 2 (CAD), mitochondrial cytochrome oxidase 1 (CO1), and elongation factor-1a (EF1a). Reference gene stability was analyzed using GeNorm, NormFinder, BestKeeper, 'Ct, and a comprehensive final ranking by RefFinder. The gene CO1 was identified as the primary reference gene since it was generally ranked in 1st or 2nd position among the tissue types containing mycetangia. The reference gene AK was identified as a secondary reference gene. In contrast, EF1a was generally ranked in the last or penultimate place for tissues containing mycetangia. Identification of two stable reference genes will aid in normalizing the expression of target genes during RT-qPCR analyses of X. germanus mycetangia.