(A) Phylogeny of newly identified Melolontha melolontha β-glucosidases and previously reported β-glucosidases of Tenebrio molitor (Tm bGlc, AF312017.1) and Chrysomela populi (Cp bGlc, KP068701.1), and myrosinases (thioglucosidases) of Phyllotreta striolata (Ps myrosinase, KF377833.1) and Brevicoryne brassicae (Bb myrosinase, AF203780.1) based on amino acid similarities using maximum likelihood method. Bootstrap values (N = 1000) are shown next to each node. Amino acid sequence alignments of the β-glucosidases are shown in Figure 3—figure supplement 1. (B) Heat map of average (n = 3) gene expression levels of M. melolontha β-glucosidases in the anterior and posterior midgut of larvae feeding on diets supplemented with water, taraxinic acid β-D-glucopyranosyl ester (TA-G), or Taraxacum officinale latex-containing diet. FPKM = fragments per kilobase of transcript per million mapped reads. (C) Activity of heterologously expressed M. melolontha β-glucosidases with TA-G, a mixture of maize benzoxazinoids, the salicinoid salicin, 4-methylsulfinylbutyl glucosinolate (4-MSOB), cellobiose, and the fluorogenic substrate 4-methylumbelliferyl-β-D-glucopyranoside (Glc-MU). Glucosidase activities of three consecutive assays with excreted proteins from insect High Five cells were measured. Negative controls (buffer, non-transfected wild-type cells, and cells transfected with green fluorescent protein) did not hydrolyze any defense metabolite. Results from the individual assays are shown in Figure 3—figure supplement 2. For deglycosylation of these compounds by M. melolontha gut protein crude extracts, refer to Figure 3—figure supplement 3. Deglycosylation assays with recombinant Mm_bGlc17 yielded highest aglycone formation; Figure 3—figure supplement 4. Raw data are available in Figure 3—source data 1.