HCN emission from <i>L. corniculatus</i> leaf macerates is strongly reduced in the highly alkaline midgut.

<p>The pH of <i>L. corniculatus</i> leaf macerates is slightly acidic (5.9±0.1 SD, N = 10, green dotted line), whereas the pH measured in the midgut lumen of <i>Z. filipendulae</i> larvae is highly alkaline (10.6±0.1 SD, N = 11, blue dotted line). HCN emission from leaf disc macerates is highest at pH 5–6, which matches the pH of <i>L. corniculatus</i> leaf macerates. However, HCN emission is significantly reduced under highly alkaline conditions at pH 10–11 present in the midgut lumen of <i>Z. filipendulae</i> larvae (one-tailed Student’s t-test, P<0.001). Each data point represents the mean (±SE) of ten independent incubations, i.e. 90 leaf discs were analysed in total.</p

β-Glucosidases from saliva and gut tissue of <i>Z. filipendulae</i> do not hydrolyse linamarin and lotaustralin.

<p><b>A.</b> Hydrolysis of cyanogenic glucosides (CNglcs) with corresponding HCN release is visualized by Feigl-Anger paper, or <b>B.</b> via fluorescence of methylumbelliferone, the hydrolysis product of the generic substrate 4-methylumbelliferyl β-D-glucopyranoside (MUG; in black). The β-glucosidases (BGDs) extracted from the saliva and gut are active enzymes as they hydrolyse MUG (<b>B.</b>), as well as prunasin in case of the gut β-glucosidase (<b>A.</b>). Importantly, the two CNglcs linamarin and lotaustralin present in the food plant <i>L. corniculatus</i> (indicated by *) are not hydrolysed by the saliva and gut β-glucosidases. Linamarin and lotaustralin are neither hydrolysed if tested individually (<b>A.</b>, top), nor hydrolysed if tested using a <i>cyd2</i> leaf macerate (<b>A.</b>, bottom). A macerate of <i>L. japonicus cyd2</i> mimics digestion of a leaf containing linamarin and lotaustralin, but does not release HCN as it lacks the corresponding BGD.</p

Saliva extracts of <i>Z. filipendulae</i> do not inhibit plant cyanogenesis.

<p>Feigl-Anger paper showing HCN emission over time from leaf macerates of <i>L. corniculatus</i> and <i>L. japonicus</i> (wild-type MG-20) incubated with either: insect saliva of <i>Z. filipendulae</i> larvae, water or heat-inactivated saliva as control (latter only on MG-20). When leaf macerates of both <i>Lotus</i> species are mixed with insect saliva, HCN emission increases at a similar rate as the leaf macerate incubated with water or heat-inactivated saliva.</p

The mandible morphology of <i>Z. filipendulae</i> enables leaf-snipping to ingest and digest large leaf fragments.

<p><b>A.</b> Larva of <i>Z. filipendulae</i> feeding on its host plant <i>L. corniculatus</i>, which contains the cyanogenic glucosides linamarin and lotaustralin. The mouthparts including the mandibles are indicated by an arrowhead. The larva is ∼ 2.5 cm long. <b>B.</b> Frontal-ventral view of the head with the two mandibles laying partly over each other. The distance between the bases of both mandibles is ∼ 600 µm (arrowheads). The leaf-processing area of the mandible is indicated by a dashed line. Both mandibles are partly covered by the labrum in a closed position. <b>C.</b> The right mandible viewed dorsally showing a round, concave and non-toothed shape with a length of ∼ 400 µm and a width of ∼ 300 µm. The leaf-processing area is indicated by a dashed line. <b>D.</b> The larval gut content shows that ingested <i>L. corniculatus</i> leaf fragments are relatively large and match the dimensions and morphology of the two mandibles. <b>E.</b> Detail of a representative <i>L. corniculatus</i> leaf fragment from the larval gut which is ∼ 550×450 µm - a similar size is retained in the frass (<b>F.</b>)<b>.</b></p