The function and evolutionary significance of a triplicated Na,K-ATPase gene in a toxin-specialized insect

Abstract

BackgroundThe 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.ResultsUsing 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).ConclusionsThese 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 materialThe online version of this article (10.1186/s12862-017-1097-6) contains supplementary material, which is available to authorized users.

Concerted evolution reveals co-adapted amino acid substitutions in Na+K+-ATPase of frogs that prey on toxic toads

“…A survey of ATP1A1 αM1-2 in toads and frogs (11) revealed a possible duplication of this gene in the toad-eating frog, Leptodactylus latrans (reported as L. ocellatus), where the resistant (R) paralog includes substitutions known to confer resistance to CGs while the sensitive (S) paralog appears to have retained the ancestral susceptibility to CGs. Neofunctionalization of ATP1A paralogs has contributed to the evolution of CG-resistance in numerous insect lineages (19)(20)(21)(22) but appear to be rare among CG-resistant vertebrates. Further, the fate of duplicated genes and the probability that they will neofunctionalize is predicted to depend on the strength of selection for functional differentiation relative to the rate of non-allelic gene conversion (NAGC), a form of nonreciprocal genetic exchange that homogenizes sequence variation between duplicated genes, thereby impeding divergence (23)(24)(25).…”

New ways to acquire resistance: imperfect convergence in insect adaptations to a potent plant toxin

“…A similar duplication of the Na,K-ATPase α1 gene was found in lygaeid bugs (Heteroptera, Lygaeidae), Chrysochus leaf beetles, the weevil Rhyssomatus lineaticollis (Coleoptera, Chrysomelidae and Curculionidae) [9] and the agromyzid fly Phytomyza hellebori (Diptera, Agromyzidae) [11]. In all cases where tissue-specific expression patterns could be established, results show that the more conserved copy is expressed in the nervous system, while the more heavily substituted copies are present in the remainder of the body but also to some extent in the nervous tissue [9,22]. Our functional tests suggest that this allows for an adjustment of the enzyme characteristics (like ion pumping efficiency versus resistance to cardiac glycosides) to the individual needs of specific tissues [20][21][22].…”

“…Mutagenesis experiments corroborate the functional importance of substitutions at these positions as their exchange leads to cardiac glycoside-resistant Na,K-ATPases [10,[16][17][18][19][20]. In several but not all species, resistance to cardiac glycosides in addition involves duplications of the Na,K-ATPase α gene [9,11], leading to paralogues that trade-off resistance versus other functions like ion pumping efficiency [20][21][22].…”

“…F786N, αM6 T979A) that result in extremely resistant Na,K-ATPases, yet at the same time, this seems to negatively affect the ion channel and comes at the expense of drastically reduced ion pumping activity [20][21][22]. On the other hand, the expression construct mimicking the single S. gregaria copy and the pyrgomorphid brain copy yielded a sensitive form of the enzyme, albeit somewhat less sensitive than the D. melanogaster wild-type.…”

Cardenolide‐defended milkweed bugs do not evoke learning in Nephila senegalensis spiders

“…These red and black colored species tolerate and take up deterrent plant chemicals (usually cardenolides) with the most famous example being the large milkweed bug Oncopeltus fasciatus (Dallas, 1852) (Bramer, Dobler, Deckert, Stemmer, & Petschenka, ; Duffey & Scudder, ; Scudder & Duffey, ; Scudder, Moore, & Isman, ; Von Euw, Reichstein, & Rothschild, ). Both the nymphs and adults of this brightly colored species obtain a variety of potentially toxic, plant produced cardenolides from Asclepias seeds, whereas species reared on sunflower seeds lack these chemicals (Duffey & Scudder, ; Lohr et al., ; Scudder et al., ). O. fasciatus possess specific morphological adaptations used for cardenolide uptake and accumulation (Bramer, Friedrich, & Dobler, ; Scudder & Meredith, ).…”