Stability of Nuclear and Mitochondrial Reference Genes in Selected Tissues of the Ambrosia Beetle Xylosandrus germanus

SIMPLE SUMMARY: The ambrosia beetle Xylosandrus germanus (Blandford) 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 of this beetle. Female X. germanus adults use a pouch-like structure (i.e., mycangium) to maintain and transport spores of their fungal mutualist. To facilitate future studies examining gene expression of X. germanus’ mycangium, 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-phosphate synthetase 2-aspartate transcarbamylase-dihydroorotase (CAD), mitochondrial cytochrome oxidase 1 (CO1), and elongation factor-1α (EF1α). The reference genes CO1 and AK were identified as primary and secondary reference genes. By contrast, EF1α 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. ABSTRACT: The fungus-farming ambrosia beetle Xylosandrus germanus (Blandford) uses a pouch-like structure (i.e., mycangium) 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 mycangium. Complementary DNA was synthesized using selected tissue types from laboratory-reared and field-collected X. germanus consisting of the whole body, head + thorax, deflated or inflated mycangium + scutellum, inflated mycangium, and thorax + abdomen. Quantitative reverse-transcription PCR reactions were performed using primers for 28S ribosomal RNA (28S rRNA), arginine kinase (AK), carbamoyl-phosphate synthetase 2-aspartate transcarbamylase-dihydroorotase (CAD), mitochondrial cytochrome oxidase 1 (CO1), and elongation factor-1α (EF1α). Reference gene stability was analyzed using GeNorm, NormFinder, BestKeeper, ΔCt, and a comprehensive final ranking by RefFinder. The gene CO1 was identified as the primary reference gene since it was generally ranked in first or second position among the tissue types containing the mycangium. Reference gene AK was identified as a secondary reference gene. In contrast, EF1α was generally ranked in the last or penultimate place. Identification of two stable reference genes will aid in normalizing the expression of target genes for subsequent gene expression studies of X. germanus’ mycangium.