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/β-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 β-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 αAβ1 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.