Bait, not reward: CO₂-enriched Nepenthes pitchers secrete toxic nectar

Nepenthes pitchers are leaf-evolved biological traps containing high levels of CO₂ within them. Extrafloral nectar (EFN) secreted by these pitchers has long been regarded as the major reward to visiting arthropods, but its chemical constituents and their role in prey capture are least explored. In this study Nepenthes EFNs were isolated, and their sugars, amino acids, proteins, vitamin C and fatty acids were analysed using HPTLC-densitometry, UFLC, and biochemical assays. C:N ratio, minerals and volatiles of EFNs were determined by CHNS, ICP-OES and headspace-GC-MS, respectively; metabolic profiling was carried out using LC-MS. Peristome/lid EFNs and their active constituent were subjected to acetylcholinesterase (AChE) inhibition assays; the AChE inhibitor was characterized by bioactivity guided isolation and spectroscopy. Here we demonstrate Nepenthes EFN as a sugar (glucose-fructose-sucrose) mix with high C:N ratio, minimal amino acids, proteins, and vitamin C. N. khasiana peristome and lid EFNs displayed strong AChE inhibition; the naphthoquinone derivative, (+)-isoshinanolone, was identified as the AChE inhibitor. Plumbagin, the major volatile naphthoquinone in Nepenthes, also showed strong AChE inhibition. Direct EFN- and (+)-isoshinanolone-feeding bioassays showed symptoms of cholinergic toxicity in ants. Nepenthes EFN is a toxic bait which hinders neuronal activity in visiting arthropods. Their pitchers adopt various deceptive strategies for prey capture, and our study abolishes the notion that Nepenthes EFN is a reward to visiting ants and other arthropods. Moreover, our findings suggest that elevated CO₂ levels within the pitchers play a crucial role in regulating the growth, metabolism, herbivory, and carnivory in Nepenthes.