Validation of reference genes for quantitative real-time PCR in tiger beetles across sexes, body parts, sexual maturity and immune challenge.

Reference genes are frequently used as normalizers for expression studies despite not being previously verified to present suitable stabilities. Considering the interest that tiger beetles have generated in the past years, resulting in a variety of studies, it is crucial to dispose of a validated reference gene panel for expression studies. Nine candidate genes were tested in Cicindela campestris and Calomera littoralis across several conditions and their transcription levels were assessed with geNorm, NormFinder, BestKeeper and ΔC method algorithms. Results showed high stabilities across sexes, immune challenge and gonad developmental stages for all genes tested, while body parts comparison presented less constant expression values. Only two genes are sufficient to perform a proper normalization for most of the conditions tested, except for the body parts comparison in C. littoralis, which requires the use of at least three reference genes. On the whole, no universal gene is found to be suitable for all situations, but according to the acceptable range of values, NADH, B-t, Vatpase and ArgKin seem to present the most constant expression stability, indicating their suitability as reference genes in most of the conditions. This is the first report evaluating the stability of housekeeping genes in adephagan beetles.