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

Abstract

Insect resistance to plant toxins is widely assumed to have evolved in response to using defended plants as a dietary resource. We tested this hypothesis in the milkweed butterflies (Danaini) which have progressively evolved higher levels of resistance to cardenolide toxins based on amino acid substitutions of their cellular sodium-potassium pump (Na(+)/K(+)-ATPase). Using chemical, physiological and caterpillar growth assays on diverse milkweeds (Asclepias spp.) and isolated cardenolides, we show that resistant Na(+)/K(+)-ATPases are not necessary to cope with dietary cardenolides. By contrast, sequestration of cardenolides in the body (as a defence against predators) is associated with the three levels of Na(+)/K(+)-ATPase resistance. To estimate the potential physiological burden of cardenolide sequestration without Na(+)/K(+)-ATPase adaptations, we applied haemolymph of sequestering species on isolated Na(+)/K(+)-ATPase of sequestering and non-sequestering species. Haemolymph cardenolides dramatically impair non-adapted Na(+)/K(+)-ATPase, but had systematically reduced effects on Na(+)/K(+)-ATPase of sequestering species. Our data indicate that major adaptations to plant toxins may be evolutionarily linked to sequestration, and may not necessarily be a means to eat toxic plants. Na(+)/K(+)-ATPase adaptations thus were a potential mechanism through which predators spurred the coevolutionary arms race between plants and insects.

Keywords: adaptation; cardenolide; coevolution; milkweed butterflies; sequestration; tritrophic interaction.

Figure 1.

Caterpillar weights after 3 days on eight species of milkweed. Caterpillars of Euploea core (grey, squares), Danaus gilippus (blue, circles) and D. plexippus (red, triangles) were reared from hatching on eight species of Asclepias. Mean cardenolide concentrations (±s.e.) in leaves are shown on the x-axis. Species codes: inc, Asclepias incarnata; syr, A. syriaca; hal, A. hallii; cor, A. cordifolia; vir, A. viridis; cur, A. curassavica; lin, A. linaria; asp, A. asperula. Inset: mean caterpillar mass (±s.e.) was not different among the three caterpillar species. (Online version in colour.)

Figure 2.

Caterpillar growth on leaf discs painted with isolated toxins. Caterpillars of E. core (grey) and D. plexippus (red) were fed A. syriaca leaf discs painted with an equimolar mixture of ouabain and digitoxin. Control, painted with methanol only; high cardenolide, 3 µg cardenolide per mg dry weight; very high cardenolide, 6 µg cardenolide per mg dry weight. Bars represent least square means ± s.e. (corrected for initial mass and plant mass consumed). (Online version in colour.)

Figure 3.

Concentration of toxins in caterpillar haemolymph. Haemolymph from caterpillars raised on eight species of Asclepias was analysed by HPLC; plant species codes are given in figure 1; plant species are arranged from lowest to highest foliar cardenolide concentration. Bars represent means ± s.e. Grey bars, Euploea core; blue bars, Danaus gilippus; red bars, D. plexippus. No cardenolides were detectable in the haemolymph of E. core (grey bars are shown for clarity). The grey bar for A. linaria is lacking as no E. core haemolymph sample was obtained. Inset: means ± s.e. of haemolymph cardenolides over all plant species (Online version in colour.)

Figure 4.

Toxic impact of sequestered cardenolides on adapted and non-adapted Na⁺/K⁺-ATPase. The impact of sequestered cardenolides on D. gilippus and D. plexippus Na⁺/K⁺-ATPase activity was assessed and compared with the effect on E. core Na⁺/K⁺-ATPase (lower activity indicates greater enzyme inhibition). Haemolymph cardenolide samples from D. gilippus (blue) and D. plexippus (red) caterpillars raised on eight Asclepias species were applied on the isolated Na⁺/K⁺-ATPase of the donor species and on the Na⁺/K⁺-ATPase of E. core in in vitro assays. Each dot represents the activity of two different butterfly Na⁺/K⁺-ATPases exposed to the same haemolymph sample. The dashed grey line represents the 1 : 1 relationship. Inset: sensitivity of E. core (IC₅₀: 1.04 × 10⁻⁶ M) (grey), D. gilippus (IC₅₀: 2.88 × 10⁻⁶ M) (blue) and D. plexippus (red) Na⁺/K⁺-ATPase (IC₅₀ 1.06 × 10⁻⁴ M) to the standard cardenolide ouabain. (Online version in colour.)

Figure 5.

Recovery of caterpillars upon toxin injection. Caterpillars of E. core (grey) and D. plexippus (red) were either injected with water (control) or the standard cardenolide ouabain (10⁻² M in water). Caterpillars which had resumed feeding by the next day were scored as recovered. Inset: typical appearance of an E. core caterpillar which is paralysed after injection with ouabain (response observed in 11 of 13 injected caterpillars). Sample size ranged from seven to 13 caterpillars. (Online version in colour.)

Figure 6.

Cardenolide concentration of caterpillar midgut contents. Grey bars, E. core; blue bars, D. gilippus; red bars, D. plexippus. Dashed horizontal lines indicate mean plant cardenolide concentration. Inset: means ± s.e. of cardenolides of gut contents over five plant species. For quantitative evaluation of cardenolides in the gut contents, we focused on the dominant peaks (see Material and methods, the trend that midgut contents of E. core have cardenolide concentrations below plant material is upheld when all cardenolide peaks detected in E. core are taken into account). (Online version in colour.)