Fig. 3. Selective excretion of plant glucosides in vivo and in situ.

a Excretion and accumulation of plant glucosides injected in beetles. Each beetle was injected with 100 nL of an equimolar mixture of five glucosides. Beetles were sampled 30 min after the injection and after 1 day feeding on Arabidopsis (n = 10 biological replicates, five beetles per replicate). Glucoside excretion was analyzed by quantifying the amount of excreted glucosides in the feces (n = 10 biological replicates, feces of five beetles per replicate). Glucoside content in beetles and feces is expressed relative to the glucoside amounts detected in beetles 30 min after injection (set to 100%). The relative accumulation and excretion of glucosides were compared using the method of generalized least squares, respectively. Bars labeled with different letters are significantly different (P < 0.05). b Excretion of plant glucosides by isolated Malpighian tubules (Ramsay assay). The dissected Malpighian tubule was placed in a droplet of saline and a mixture of eight different plant glucosides each at a concentration of 6.7 mM. To visualize excretion, we added 0.1% (w/v) amaranth. After 2–3 h, bathing saline, Malpighian tubule, and excretion fluid were sampled, extracted, and analyzed by LC-MS/MS. Paired two-tailed Student’s t tests were used to compare the relative composition of plant glucosides in bathing saline and Malpighian tubule, and Malpighian tubule and excretion fluid, respectively. Dashed lines indicate significant differences between samples (FDR-corrected P < 0.05, n = 11 biological replicates, one dissected tubule per replicate). Data are shown as mean ± s.e.m. P values are provided in Supplementary Data 6.