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 are specialised on Ranunculaceae plants from which the larvae can sequester furostanol saponins into the haemolymph, mainly (25 )-26-[(α-L-rhamnopyranosyl)oxy]-22α-methoxyfurost-5-en-3β-yl- -β-D-glucopyranosyl-(1→3)- -[6-acetyl-β-D-glucopyranosyl-(1→3)]- -β-D-glucopyranoside (compound ). 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 L. ( species A) and L. ( species B). Larvae of species B were mostly feeding on the squares treated with the -butanol fraction from , compound being its primary non-nutritional stimulant. In contrast, all fractions stimulated feeding, with -hexane marginally more active. β-sitosterol within -hexane was determined as the nutritional stimulant. Quantitative analyses demonstrated that leaves of but not contain the ecdysteroids 20-hydroxyecdysone and polypodine B. Moreover, the haemolymph of species B larvae reared on 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 to 2.23 × 10 µmol total ecdysteroids/g fresh weight of leaves), suggesting bioaccumulation. The larvae of both species fed on 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 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 species feeding on sequester phytoecdysteroids into the larval haemolymph in the form of glycosides. In addition, compound possesses defensive and phagostimulant activities, and we present evidence for a combined effect of furostanol saponins and ecdysteroids as repellents against ants.