Escalation by duplication: Milkweed bug trumps Monarch butterfly

The iconic Monarch butterfly is probably the best‐known example of chemical defence against predation, as pictures of vomiting naive blue jays in countless textbooks vividly illustrate. Larvae of the butterfly take up toxic cardiac glycosides from their milkweed hostplants and carry them over to the adult stage. These compounds (cardiotonic steroids, including cardenolides and bufadienolides) inhibit the animal transmembrane sodium‐potassium ATPase (Na,K‐ATPase), but the Monarch enzyme resists this inhibition thanks to amino acid substitutions in its catalytic alpha‐subunit. Some birds also have substitutions and can feast on cardiac glycoside‐sequestering insects with impunity. A flurry of recent work has shown how the alpha‐subunit gene has been duplicated multiple times in separate insect lineages specializing in cardiac glycoside‐producing plants. In this issue of Molecular Ecology, Herbertz et al. toss the beta‐subunit into the mix, by expressing all nine combinations of three alpha‐ and three beta‐subunits of the milkweed bug Na,K‐ATPase and testing their response to a cardenolide from the hostplant. The findings suggest that the diversification and subfunctionalization of genes allow milkweed bugs to balance trade‐offs between resistance towards sequestered host plant toxins that protect the bugs from predators, and physiological costs in terms of Na,K‐ATPase activity.

The iconic Monarch butterfly is probably the best-known example of chemical defence against predation, as pictures of vomiting naive blue jays in countless textbooks vividly illustrate.Larvae of the butterfly take up toxic cardiac glycosides from their milkweed hostplants and carry them over to the adult stage.These compounds (cardiotonic steroids, including cardenolides and bufadienolides) inhibit the animal transmembrane sodium-potassium ATPase (Na,K-ATPase), but the Monarch enzyme resists this inhibition thanks to amino acid substitutions in its catalytic alpha-subunit.Some birds also have substitutions and can feast on cardiac glycoside-sequestering insects with impunity.A flurry of recent work has shown how the alpha-subunit gene has been duplicated multiple times in separate insect lineages specializing in cardiac glycoside-producing plants.In this issue of Molecular Ecology, Herbertz et al. toss the beta-subunit into the mix, by expressing all nine combinations of three alpha-and three beta-subunits of the milkweed bug Na,K-ATPase and testing their response to a cardenolide from the hostplant.The findings suggest that the diversification and subfunctionalization of genes allow milkweed bugs to balance trade-offs between resistance towards sequestered host plant toxins that protect the bugs from predators, and physiological costs in terms of Na,K-ATPase activity.

K E Y W O R D S
adaptation, coevolution, ecological genetics, insects, invertebrates, molecular evolution was found in the large milkweed bug, Oncopeltus fasciatus, which possesses four α-subunit paralogues (Yang et al., 2019).The underlying gene duplication events probably already occurred in the ancestor of milkweed bugs (subfamily Lygaeinae, about 600 species), which are closely associated with cardenolide-containing Apocyanaceae (Bramer et al., 2015;Zhen et al., 2012).The physically associated β-subunit, not homologous to the α-subunit but essential as a cofactor or chaperone, is also duplicated in many insects including the milkweed bug, but specific amino acid substitutions have not been studied.Although the evolutionary order of appearance of the different α-subunits in milkweed bugs and other insects can be inferred from sequence similarity, this is not the case for the β-subunits, which have duplicated much earlier and thus independently of the α-subunits in insects.
Phylogenetic analyses revealed a stepwise increase in the number of amino acid substitutions in the α-subunit paralogues of the milkweed bugs (Yang et al., 2019;Zhen et al., 2012) (Figure 1).Based on studies with recombinant ATPα-subunits of Drosophila melanogaster mimicking the four different α-subunit paralogues, this pattern was proposed to result in increased cardenolide-resistance at the expense of Na,K-ATPase activity (Dalla & Dobler, 2016).In fact, while this early study provided strong evidence that ATPα gene duplications can mitigate trade-offs between cardenolide resistance and Na,K-ATPase function in the milkweed bug, it did not fully capture the complexity of the natural system.In addition to four paralogues of the α-subunit, the milkweed bug genome encodes at least three paralogues of the associated β-subunit of the Na,K-ATPase, which also influences Na,K-ATPase activity, although to a lesser extent than the α-subunit.More importantly, the previous functional characterization was based on ouabain, a cardenolide that is not found in the milkweed bugs' host plants and thus does not provide insight into the specific interactions between host plant cardenolides and the different forms of Na,K-ATPase.
In this study, Herbertz et al. heterologously expressed three αsubunit paralogues and three β-subunit paralogues from the large milkweed bug in all nine possible combinations using the baculovirus system in insect cells and compared their activities and sensitivities towards different cardenolides.Each of these α/β combinations was more resistant towards cardenolides than the Na,K-ATPase of the monarch butterfly, a result that is consistent with inhibition assays performed with brain tissue extracts of both species (e.g.Agrawal et al., 2022).As expected, the identity of the α-subunit had a much stronger impact on kinetic parameters than the identity of the βsubunit, which nevertheless influenced both activity and sensitivity in vitro (Figure 1).Consistent with earlier work by Dalla and Dobler (2016), complexes containing the cardenolide-sensitive αC were more active than the phylogenetically derived and cardenolideresistant αA-and αB-containing complexes.Among the three αC combinations, αCβ3 was the most active in vitro (Figure 1).This barrier (Herbertz et al., 2022;et al., 2017).However, Herbertz et al. also found several striking differences between the biochemical properties of the ATPα mimics (Dalla & Dobler, 2016) and the ATPα subunits tested in this study, highlighting the importance of studying the original enzymes.
To compare the sensitivity of α/β complexes, Herbertz et al. tested two structurally different cardenolides, the commercially available standard cardenolide ouabain, and calotropin, which was isolated from larvae of the monarch butterfly.Only the latter cardenolide is found in host plants of the large milkweed bug, making it particularly valuable for the biochemical characterization of recombinant Na,K-ATPases in this study.Indeed, the α/β complexes differed drastically in their insensitivity to these two cardenolides: All αC complexes were more strongly inhibited by calotropin than by ouabain, whereas αA and αB complexes were similarly insensitive to both cardenolides, except for the αAβ1 complex, which was even more resistant to calotropin compared with ouabain (Figure 1d).
The much higher resistance of the phylogenetically derived αA-and αB-subunits to calotropin, which is fine-tuned by the associated βsubunit, shows that repeated gene duplications allowed milkweed bugs to adapt to specific toxins in their host plants.
The results of this study thus provide evidence for a coevolutionary escalation scenario in which plants diversify their chemical defences against adapted herbivores, followed by molecular adaptations to the novel defence in the herbivore.This and another recent study suggest that the evolution of more potent inhibitory cardenolides in plants is countered by at least two different mechanisms in the large milkweed bug: (1) the duplication and subfunctionalization of the target gene and (2) the ability to convert toxic cardenolides into non-toxic forms that can be sequestered without ill effects (Agrawal et al., 2022;Herbertz et al., 2023).
complex is known to be localized in the nervous tissue of O. fasciatus, where it is largely protected from cardenolides by the blood-brain F I G U R E 1 Na,K-ATPase activity and cardenolide sensitivity depend on the combination of ATPα and ATPβ paralogues in of the milkweed bug Oncopeltus fasciatus.(a) Oncopeltus fasciatus (photograph: Katja Schulz from Washington, D.C., USA, CC BY 2.0).(b) Amino acid substitutions at positions implicated in cardenolide binding in four ATPα paralogues of O. fasciatus.Amino acids of the cardenolide-sensitive ATPα from the fruit fly, Drosophila melanogaster are shown as a reference.Amino acid substitutions shown in red font were previously introduced in D. melanogaster ATPα using site-directed mutagenesis (Dalla & Dobler, 2016).(c) Structures of the host plant cardenolide calotropin and the commercially available standard cardenolide ouabain.(d) Overview of the comparative biochemical characterization of nine different α/β complexes from O. fasciatus.This study compared the basal Na,K-ATPase activity in the absence of cardenolides, and sensitivity towards calotropin and ouabain.=, similar sensitivity to both calotropin and ouabain; C, Calotropin; O, Ouabain.