Abstract
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 ΔCTmethod 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.
Highlights
Evaluating changes in gene expression has lately been one of the most frequent studies to analyse the effect of physiological responses and the biological differences among populations
Nine candidate reference genes were evaluated across different conditions in Cicindela campestris and Calomera littoralis in this study, including Arginine Kinase (ArgKin), Myosin regulatory light chain 2 (Myo), Myosin light chain alkali (Myo-Alc), 60 s acidic ribosomal p0 (60Sa), Ribosomal protein S18 (RPS18), Tubulin beta-1 chain (B-t), Vatype proton atpase 16kda proteolipid subunit (Vatpase), NADH dehydrogenase (NADH) and 40 s ribosomal protein s11 (RPS11)
Nine genes were identified as putative reference genes according to blast searches from both libraries: (i) Arginine Kinase (ArgKin), Myosin regulatory light chain 2 (Myo), Myosin light chain alkali (Myo-Alc) and 60 s acidic ribosomal p0 (60Sa) were obtained from C. littoralis; (ii) Ribosomal protein S18 (RPS18), Tubulin beta-1 chain (B-t), Vatype proton atpase 16kda proteolipid subunit (Vatpase), NADH dehydrogenase (NADH) and 40 s ribosomal protein s11 (RPS11) were identified from C. campestris
Summary
Evaluating changes in gene expression has lately been one of the most frequent studies to analyse the effect of physiological responses and the biological differences among populations. Cicindelids, commonly known as tiger beetles, are one of the most important non-pest species within the suborder Adephaga (Coleoptera), with more than 2500 species described[29] They are worldwide distributed with the exception of Tasmania, Antarctica and some remote Oceanic islands, occupying a wide range of habitats[30,31], and are considered to be good bioindicators due to their biological and evolutionary characteristics[30,32,33,34]. The availability of a selection of validated reference genes for genomic and expression analyses will allow us to obtain more reliable results in future studies. The aims of this research are (i) to examine the expression stability of different housekeeping genes in non-model beetle species across sexes, body parts, gonad developmental stages (immature/mature) and immune challenge (ii) to provide a selection of accurate reference genes for future genomic and expression analyses in cicindelids
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