Prieto Jose M, Barker Alison M, Schaffner Urs, Quetin-Leclercq Joëlle, Braca Alessandra, Boevé Jean-Luc
Department of Pharmacy, University of Pisa, 56126 Pisa, Italy.
CABI Switzerland, 2800 Delémont, Switzerland.
Plants (Basel). 2024 Aug 12;13(16):2230. doi: 10.3390/plants13162230.
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.
叶蜂属的叶蜂种类专门以毛茛科植物为食，其幼虫能够将呋甾烷醇皂苷隔离到血淋巴中，主要是(25)-26-[(α-L-鼠李糖基)氧基]-22α-甲氧基呋甾-5-烯-3β-基--β-D-吡喃葡萄糖基-(1→3)--[6-乙酰基-β-D-吡喃葡萄糖基-(1→3)]--β-D-吡喃葡萄糖苷（化合物 ）。在这项工作中，进行了薄层色谱法（TLC）、气相色谱-质谱联用（GC-MS）和高效液相色谱-二极管阵列检测-电喷雾电离质谱联用（HPLC-DAD-ESI/MS）分析，并结合取食、重复模拟攻击和蚂蚁威慑生物测定，以扩展关于两种专门以毛茛属植物为食的叶蜂种类（物种A和物种B）的化学生态学知识。物种B的幼虫大多取食经石龙芮正丁醇部位处理过的叶片，化合物 是其主要的非营养性刺激物。相比之下，所有的部位都刺激取食，正己烷的活性略高。正己烷中的β-谷甾醇被确定为营养性刺激物。定量分析表明，石龙芮的叶片含有蜕皮甾酮20-羟基蜕皮酮和多足甾酮B，而毛茛的叶片则不含。此外，以石龙芮饲养的物种B幼虫的血淋巴中含有多足甾酮B和20-羟基蜕皮酮的糖苷，浓度为2.5至6.8微摩尔/克血淋巴鲜重。该浓度比其寄主植物中苷元的浓度范围（3.63×10至2.23×10微摩尔总蜕皮甾酮/克叶片鲜重）高数千倍，表明存在生物积累。以毛茛为食的两种叶蜂幼虫的血淋巴中均未显示出任何蜕皮甾酮的痕迹，这表明这些化合物在其防御中起兼性作用，并且它们无法内源性合成这些化合物。含有蜕皮甾酮的血淋巴对毛蚁属蚂蚁工蚁（它们的天然捕食者之一）在浓度为0.8毫克/毫升时具有显著的取食威慑作用。在第1天和第2天的模拟攻击之间，幼虫保持了有效的糖基化蜕皮甾酮威慑水平（约175毫摩尔），但在第3天水平明显下降（约75毫摩尔）。大多数幼虫（89%）在第一次攻击中存活，但只有23%在连续的第二次攻击中存活。总之，我们首次报告，两种以毛茛为食的叶蜂物种将植物蜕皮甾酮以糖苷的形式隔离到幼虫血淋巴中。此外，化合物 具有防御和摄食刺激活性，并且我们提供了呋甾烷醇皂苷和蜕皮甾酮作为驱蚁剂的联合作用的证据。
