Coevolutionary escalation led to differentially adapted paralogs of an insect's Na,K-ATPase optimizing resistance to host plant toxins.

Cardiac glycosides are chemical defence toxins known to fatally inhibit the Na,K-ATPase (NKA) throughout the animal kingdom. Several animals, however, have evolved target-site insensitivity through substitutions in the otherwise highly conserved cardiac glycoside binding pocket of the NKA. The large milkweed bug, Oncopeltus fasciatus, shares a long evolutionary history with cardiac glycoside containing plants that led to intricate adaptations. Most strikingly, several duplications of the bugs' NKA1α gene provided the opportunity for differential resistance-conferring substitutions and subsequent sub-functionalization of the enzymes. Here, we analysed cardiac glycoside resistance and ion pumping activity of nine functional NKA α/ß-combinations of O. fasciatus expressed in cell culture. We tested the enzymes with two structurally distinct cardiac glycosides, calotropin, a host plant compound, and ouabain, a standard cardiac glycoside. The identity and number of known resistance-conferring substitutions in the cardiac glycoside binding site significantly impacted activity and toxin resistance in the three α-subunits. The ß-subunits also influenced the enzymes' characteristics, yet to a lesser extent. Enzymes containing the more ancient αC-subunit were inhibited by both compounds but much more strongly by the host plant toxin calotropin than by ouabain. The sensitivity to calotropin was diminished in enzymes containing the more derived αB and αA, which were only marginally inhibited by both cardiac glycosides. This trend culminated in αAß1 having higher resistance against calotropin than against ouabain. These results support the coevolutionary escalation of plant defences and herbivore tolerance mechanisms. The possession of multiple paralogs additionally mitigates pleiotropic effects by compromising between ion pumping activity and resistance.

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

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.

Community-wide convergent evolution in insect adaptation to toxic cardenolides by substitutions in the Na,K-ATPase.

The extent of convergent molecular evolution is largely unknown, yet is critical to understanding the genetics of adaptation. Target site insensitivity to cardenolides is a prime candidate for studying molecular convergence because herbivores in six orders of insects have specialized on these plant poisons, which gain their toxicity by blocking an essential transmembrane carrier, the sodium pump (Na,K-ATPase). We investigated gene sequences of the Na,K-ATPase α-subunit in 18 insects feeding on cardenolide-containing plants (spanning 15 genera and four orders) to screen for amino acid substitutions that might lower sensitivity to cardenolides. The replacement N122H that was previously shown to confer resistance in the monarch butterfly (Danaus plexippus) and Chrysochus leaf beetles was found in four additional species, Oncopeltus fasciatus and Lygaeus kalmii (Heteroptera, Lygaeidae), Labidomera clivicollis (Coleoptera, Chrysomelidae), and Liriomyza asclepiadis (Diptera, Agromyzidae). Thus, across 300 Myr of insect divergence, specialization on cardenolide-containing plants resulted in molecular convergence for an adaptation likely involved in coevolution. Our screen revealed a number of other substitutions connected to cardenolide binding in mammals. We confirmed that some of the particular substitutions provide resistance to cardenolides by introducing five distinct constructs of the Drosophila melanogaster gene into susceptible eucaryotic cells under an ouabain selection regime. These functional assays demonstrate that combined substitutions of Q(111) and N(122) are synergistic, with greater than twofold higher resistance than either substitution alone and >12-fold resistance over the wild type. Thus, even across deep phylogenetic branches, evolutionary degrees of freedom seem to be limited by physiological constraints, such that the same molecular substitutions confer adaptation.

Substitutions in the cardenolide binding site and interaction of subunits affect kinetics besides cardenolide sensitivity of insect Na,K-ATPase.

Substitutions within the cardenolide target site of several insects' Na,K-ATPase α-subunits may confer resistance against toxic cardenolides. However, to which extent these substitutions alter the Na,K-ATPase's kinetic properties and how they interact with different ß-subunits is not clear. The cardenolide-adapted milkweed bug Oncopeltus fasciatus possesses three paralogs of the α-subunit (A, B, and C) that differ in number and identity of resistance-conferring substitutions. We introduced these substitutions into the α-subunit of Drosophila melanogaster and combined them with the ß-subunits Nrv2.2 and Nrv3. The substitutions Q111T-N122H-F786N-T797A (A-copy mimic) and Q111T-N122H-F786N (B-copy mimic) mediated high insensitivity to ouabain, yet they drastically lowered ATPase activity. Remarkably, the identity of the ß-subunit was decisive and all α-subunits were less active when combined with Nrv3 than when combined with Nrv2.2. Both the substitutions and the co-expressed ß-subunit strongly affected the enyzme's affinity for Na+ and K+. Na+ affinity was considerably higher for all enzymes expressed with nrv3 while expression with nrv2.2 mostly increased K+ affinity. Our results provide the first evidence that resistance against cardenolides comes at the cost of significantly altered kinetic properties of the Na,K-ATPase. The ß-subunit can strongly modulate these properties but cannot fully compensate for the effect of the substitutions.

Gene duplications circumvent trade-offs in enzyme function: Insect adaptation to toxic host plants.

Herbivorous insects and their adaptations against plant toxins provide striking opportunities to investigate the genetic basis of traits involved in coevolutionary interactions. Target site insensitivity to cardenolides has evolved convergently across six orders of insects, involving identical substitutions in the Na,K-ATPase gene and repeated convergent gene duplications. The large milkweed bug, Oncopeltus fasciatus, has three copies of the Na,K-ATPase α-subunit gene that bear differing numbers of amino acid substitutions in the binding pocket for cardenolides. To analyze the effect of these substitutions on cardenolide resistance and to infer possible trade-offs in gene function, we expressed the cardenolide-sensitive Na,K-ATPase of Drosophila melanogaster in vitro and introduced four distinct combinations of substitutions observed in the three gene copies of O. fasciatus. With an increasing number of substitutions, the sensitivity of the Na,K-ATPase to a standard cardenolide decreased in a stepwise manner. At the same time, the enzyme's overall activity decreased significantly with increasing cardenolide resistance and only the least substituted mimic of the Na,K-ATPase α1C copy maintained activity similar to the wild-type enzyme. Our results suggest that the Na,K-ATPase copies in O. fasciatus have diverged in function, enabling specific adaptations to dietary cardenolides while maintaining the functionality of this critical ion carrier.

Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals.

Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase α-subunit of Drosophila melanogaster together with the ß-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC50 for the single mutations of N122H (61.0 µM) or T797A (63.3 µM) was increased roughly 250-fold relative to the wild-type (0.24 µM), the double mutations of Q111V-N122H (IC50 550 µM) and Q111T-N122H (IC50 583 µM) proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on cardenolide exposed insects has resulted in very efficient substitutions that decrease cardenolide sensitivity extremely.