Furostanol Saponins and Ecdysteroids from Plants of the Genus Helleborus as Phagostimulants and Predator Deterrents for Larvae of Two Monophadnus Sawfly Species

Sawfly species of the genus <i>Monophadnus</i> are specialised on Ranunculaceae plants from which the larvae can sequester furostanol saponins into the haemolymph, mainly (25<i>R</i>)-26-[(α-L-rhamnopyranosyl)oxy]-22α-methoxyfurost-5-en-3β-yl-<i>O</i>-β-D-glucopyranosyl-(1→3)-<i>O</i>-[6-acetyl-β-D-glucopyranosyl-(1→3)]-<i>O</i>-β-D-glucopyranoside (compound <b>1</b>). In this work, TLC, GC-MS, and HPLC-DAD-ESI/MS analyses together with feeding, repeated simulated attacks, and ant deterrence bioassays were conducted to extend the chemoecological knowledge about two sawfly species specialised on <i>H. foetidus</i> L. (<i>Monophadnus</i> species A) and <i>H. viridis</i> L. (<i>Monophadnus</i> species B). Larvae of <i>Monophadnus</i> species B were mostly feeding on the squares treated with the <i>n</i>-butanol fraction from <i>H. foetidus</i>, compound <b>1</b> being its primary non-nutritional stimulant. In contrast, all <i>H. viridis</i> fractions stimulated feeding, with <i>n</i>-hexane marginally more active. β-sitosterol within <i>n</i>-hexane was determined as the nutritional stimulant. Quantitative analyses demonstrated that leaves of <i>H. viridis</i> but not <i>H. foetidus</i> contain the ecdysteroids 20-hydroxyecdysone and polypodine B. Moreover, the haemolymph of <i>Monophadnus</i> species B larvae reared on <i>H. viridis</i> contained the glycosides of polypodine B and 20-hydroxyecdysone at a concentration of 2.5 to 6.8 µmol/g fresh weight of haemolymph. This concentration is several thousand times higher than the concentration range of the aglycones in their host plant (3.63 × 10^-4 to 2.23 × 10^-4 µmol total ecdysteroids/g fresh weight of leaves), suggesting bioaccumulation. The larvae of both species fed on <i>H. foetidus</i> do not show any traces of ecdysteroids in their haemolymph, indicating a facultative role of these compounds in their defence as well as their inability to endogenously synthesise these compounds. The haemolymph containing ecdysteroids was a significant feeding deterrent against <i>Myrmica rubra</i> L. ant workers (one of their natural predators) at 0.8 mg/mL. The larvae kept effective deterrent levels of glycosylated ecdysteroids (≅175 mM) between simulated attacks on days 1 and 2, but the levels clearly decreased on day 3 (≅75 mM). Most larvae (89%) survived a first attack but only 23% a consecutive second one. As a conclusion, we report for the first time that two <i>Monophadnus</i> species feeding on <i>H. viridis</i> sequester phytoecdysteroids into the larval haemolymph in the form of glycosides. In addition, compound <b>1</b> possesses defensive and phagostimulant activities, and we present evidence for a combined effect of furostanol saponins and ecdysteroids as repellents against ants.