STEPWISE EVOLUTION OF RESISTANCE TO TOXIC CARDENOLIDES VIA GENETIC SUBSTITUTIONS IN THE NA⁺/K⁺-ATPASE OF MILKWEED BUTTERFLIES (LEPIDOPTERA: DANAINI)

Abstract

Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed

Na+/K+-ATPase resistance and cardenolide sequestration: basal adaptations to host plant toxins in the milkweed bugs (Hemiptera: Lygaeidae: Lygaeinae)

“…Na þ /K þ -ATPase assays were performed as described in Petschenka et al [31]. Briefly, brains and thoracic ganglia of hemipteran specimens (killed and stored at 2808C) were dissected under deionized water, pooled (see table 1 for numbers of individuals used) and homogenized in deionized water (500 ml) using an all glass grinder (Wheaton).…”

“…Cardenolide sensitivity of Na þ /K þ -ATPase was determined by photometric quantification of inorganic phosphate released from ATP by Na þ /K þ -ATPase at different concentrations of ouabain over a period of 20 min at 378C. To test for linearity, we measured a time course of P i release over the period of incubation (reactions stopped after 0, 5, 10, 15 and 20 min) using a Na þ /K þ -ATPase preparation of O. fasciatus under all reaction conditions (10 23 to 10 28 M plus controls) and found that P i release was always linear (see [31] and the electronic supplementary material, figure S2). Linearity under all conditions ensures that ouabain inhibition curves are not biased by nonlinear P i release over time under different incubation regimes.…”

Milkweed butterfly resistance to plant toxins is linked to sequestration, not coping with a toxic diet

“…Despite the widespread appreciation of sequestration as an extreme component of host plant specialization and adaptation against predation [9,10], the mechanisms of how sequestration integrates into coevolutionary theory [3,11] are barely understood. To test the relative importance of coping with a toxic diet versus sequestration of toxins in shaping insect adaptations, here we assess the functional consequences of an evolutionarily escalating resistance trait across the milkweed butterflies [12,13]. Cardenolides (cardiac glycosides) are highly potent plant toxins that specifically inhibit Na þ /K þ -ATPase, an essential animal cation transporter [14,15].…”

“…Among the 16 species of Danaini whose Na þ /K þ -ATPase genes were sequenced and Na þ /K þ -ATPases physiologically assayed, resistance to cardenolides was determined by three discrete molecular forms [12].…”

“…Here we report on studies of three representative species of milkweed butterflies, each having one of these different forms of Na þ /K þ -ATPase [12], ranging from sensitive to highly resistant to cardenolides. Caterpillars of the common crow (Euploea core; sensitive Na þ /K þ -ATPase), the queen butterfly (Danaus gilippus; intermediate resistance Na…”