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Despite its history as a developmental and evolutionary model organism, gene expression analysis in the large milkweed bug, Oncopeltus fasciatus, has rarely been explored using quantitative real-time PCR. The strength of this method depends greatly on the endogenous controls used for normalization, which are lacking for the milkweed bug system. Here, to fill in this gap in our knowledge, we validated the stability of a set of ten candidate reference genes identified from the O. fasciatus transcriptome, and did so upon exposure to a dietary toxin, a cardiac glycoside, and across four different exposure periods. To increase robustness against gDNA contaminants, genome resources were used to design intron-bridging primers. A comprehensive stability validation by the Bestkeeper, Normfinder, geNorm and comparative ΔCt methods identified ef1a and tubulin as the most stable genes across treatments and time points, whereas 18S rRNA was the most unstable. However, accounting for the temporal scale indicated that time point confined normalizers might enable higher quantification accuracy for treatment comparison. Overall this study demonstrates: (i) a robust RT-qPCR primer design approach is possible for non-model organisms where genome annotation is often incomplete, and (ii) the importance of detailed reference gene stability exploration in multifactorial experimental designs.

Background The Na,K-ATPase is a vital animal cell-membrane protein that maintains the cell’s resting potential, among other functions. Cardenolides, a group of potent plant toxins, bind to and inhibit this pump. The gene encoding the α-subunit of the pump has undergone duplication events in some insect species known to feed on plants containing cardenolides. Here we test the function of these duplicated gene copies in the cardenolide-adapted milkweed bug, Oncopeltus fasciatus, which has three known copies of the gene: α1A, α1B and α1C. Results Using RT-qPCR analyses we demonstrate that the α1C is highly expressed in neural tissue, where the pump is generally thought to be most important for neuron excitability. With the use of in vivo RNAi in adult bugs we found that α1C knockdowns suffered high mortality, where as α1A and α1B did not, supporting that α1C is most important for effective ion pumping. Next we show a role for α1A and α1B in the handling of cardenolides: expression results find that both copies are primarily expressed in the Malpighian tubules, the primary insect organ responsible for excretion, and when we injected either α1A or α1B knockdowns with cardenolides this proved fatal (whereas not in controls). Conclusions These results show that the Na,K-ATPα gene-copies have taken on diverse functions. Having multiple copies of this gene appears to have allowed the newly arisen duplicates to specialize on resistance to cardenolides, whereas the ancestral copy of the pump remains comparatively sensitive, but acts as a more efficient ion carrier. Interestingly both the α1A and α1B were required for cardenolide handling, suggesting that these two copies have separate and vital functions. Gene duplications of the Na,K-ATPase thus represent an excellent example of subfunctionalization in response to a new environmental challenge. Electronic supplementary material The online version of this article (10.1186/s12862-017-1097-6) contains supplementary material, which is available to authorized users.

Evolution of insensitivity to the toxic effects of cardiac glycosides has become a model in the study of convergent evolution, as five taxonomic orders of insects use the same few similar amino acid substitutions in the otherwise highly conserved Na,K-ATPase α. We show here that insensitivity in pyrgomorphid grasshoppers evolved along a slightly divergent path. As in other lineages, duplication of the Na,K-ATPase α gene paved the way for subfunctionalization: one copy maintains the ancestral, sensitive state, while the other copy is resistant. Nonetheless, in contrast with all other investigated insects, the grasshoppers' resistant copy shows length variation by two amino acids in the first extracellular loop, the main part of the cardiac glycoside-binding pocket. RT-qPCR analyses confirmed that this copy is predominantly expressed in tissues exposed to the toxins, while the ancestral copy predominates in the nervous tissue. Functional tests with genetically engineered Drosophila Na,K-ATPases bearing the first extracellular loop of the pyrgomorphid genes showed the derived form to be highly resistant, while the ancestral state is sensitive. Thus, we report convergence in gene duplication and in the gene targets for toxin insensitivity; however, the means to the phenotypic end have been novel in pyrgomorphid grasshoppers.